light microscopy in biology: a practical approach (the practical approach series) (2nd edition)

Light Microscopy in Biology

The Practical Approach Series

SERIES EDITOR

B. D. HAMESDepartment of Biochemistry and Molecular Biology

University of Leeds, Leeds LS2 9JT, UK

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Light Microscopy inBiology

Second Edition

A Practical Approach

Edited by

ALAN J. LACEYDepartment of Biology and Biochemistry, Brunei,

The University of West London, Uxbridge, Middlesex UB8 3PH, UK

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Preface

Preparing a new edition of an originally successful first edition must alwaysresult in a choice having to be made as to what to leave out to make room forany new material. Optical microscopy has developed very fast in the last tenyears and yet the same principles of achieving a good image by the interactionof light with the matter under investigation are still there. Developing tech-niques in confocal and near-field microscopy have been given chapters as havecalcium ion and pH imaging which reflect something of the enormous develop-ment of fluorochromes and fluorescent microscopy. Video cameras and videorecorders used to record images for visual examination or to lead on to imageanalysis by subsequent processing in digital or other format have all madelarge contributions to the development of optical microscopy.

Basic optical microscopy in which aspects of resolution of fine detail and therequirement of contrast to make that small detail visible has been retained.The recording of the image by wet chemistry methods is still a routine require-ment in many projects but the use of video cameras and printers is beginningto challenge the dark-room photochemical production of still images. Thebasic immunohistochemistry and that of differential staining particularly in thepreparation of tissues and chromosomes has been retained while new empha-sis has been given to such topics as fluoroprobes for calcium and pH imagingand microinjection of materials into living cells.

The use of optical microscopy techniques such as near-field and nanovidto push the resolution beyond the traditionally accepted diffraction limitattempts to bridge the gap between optical and electron microscopy. Surfacedetails of living cells are very important components in the study of drugreaction and these together with cells to surface contacts are assisted by thetechniques of reflection-contrast microscopy and evanescent illumination.

The editor wishes to thank all the many colleagues, and particularly DrPeter Hobson, for their patience in technical discussions. Mr D. J. Thomson isgratefully acknowledged for his help in many practical ways. Thanks are due tothe production team of OUP who have been so helpful in preparing this bookover a long period.

Oxford A. J. L.Jan 1999

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Contents

List of Contributors xixAbbreviations xxi

1. Basic optical microscopy 1A. J. Lacey

1. Introduction 1

2. The microscope and its use 1

3. Summary of the process of image formation in themicroscope 3

Lamp collector lens and condensers 3

4. Kohler illumination 7

5. Resolution in the microscope 10

6. Magnification 14

7. Interim summary 14

8. Contrast methods 16Bright-field 16Dark-field 17Phase-contrast 23Differential interference contrast (DIC) 25Fluorescence microscopy 26Provisional summary of contrast techniques 28Summary of contrast techniques 29

9. Recording the image 29Microscope/camera attachments 30Summary 37

Acknowledgements 43

References 43

2. Introduction to confocal microscopy 45P. J. Shaw

1. Introduction 45

2. The problem of out-of-focus light 46The confocal principle: explanation by ray optics 47

Contents

Linear, shift-invariant imaging and the point spread function 48The shape of the point spread function 49Aberrations and the limits to linear, shift-invariant imaging 51

J. Practical implementation of confocal scanning systems 54Point scanning 54Slit scanning 55Spinning disc 56Two-photon imaging 57

1. Comparison of conventional, wide-field fluorescenceimaging with confocal fluorescence imaging 58

Noise and resolution 58Out-of-focus light 60When should confocal microscopy be used? 61

5. Practical examples of specimen preparation for confocalimaging 63

References 70

3. Video microscopy 73Dieter G. Weiss, Willi Maile, Robert A. Wick, and Walter Steffen

1. Video microscopy and the equipment required 73Introduction 73General strategies of electronic image improvement 77The different video-microscopic techniques 78Electronic equipment for video microscopy 89Considerations on the microscope 101

2. High resolution: video-enhanced contrast microscopy 106Different types of video-enhanced contrast microscopy 106Sample preparation 108Procedure for image generation 110Interpretation 116Typical applications and limitations 117

3. Low light: video-intensified microscopy 123Introduction 123Procedure for image generation 123Typical applications 127

4. Image analysis: video-based techniques for measurements inliving cells 130

Spatial measurements and motion analysis 131Intensity measurements 132

Contents

5. Documentation and presentation of video microscopy data 133Video recording 133Obtaining printouts for presentation and publication 141Preparing and presenting video sequences 143

Acknowledgements 146

References 146

Further reading 148

4. Microscopy of chromosomes 151A. T. Sumner and A. R. Leitch

1. Introduction 151

2. Methods of preparing chromosomes 151Routine preparation of mammalian chromosomes 152Preparation of cells by cytocentrifugation for immunocytochemical

studies of chromosomes 155Preparation of chromosomes from plant cells 156Assessment of the quality of chromosome preparations 159

3. Uniform (solid) staining of chromosomes 160

4. Chromosome banding 161The classification of chromosome bands 161C-banding 162G-banding 164Ag-NOR staining for nucleolus organizing regions 164CREST labelling of kinetochores 166

5. In situ hybridization 168Probe preparation 170In situ hybridization reaction 173

6. Observation and recording of images of chromosomes 177Observation of banding with absorbing dyes 178Observation of fluorescent chromosomes 178Photography of chromosomes 180Other methods of image capture 181

References 183

5. Immunohistochemistry 185Michael G. Ormerod and Susanne F. Imrie

1. Introduction 185

2. Antibodies 186

xi

Contents

Immunoglobulin structure 186Polyclonal antisera 187Monoclonal antibodies 187Purification of antibodies 188Specificity of antibody reactions 188Storage of antibodies 189

3. Effect of tissue processing on antigens 189Choosing conditions for processing 189Revealing hidden antigens 192

4. Choice of label 194Fluorescent labels 194Enzymatic labels 194Colloidal gold 197Selecting a label 197

5. Methods of application 197The direct method 198The indirect method 198Enzyme-anti-enzyme methods 199Systems using biotin-avidin 200Other methods 201

6. Experimental methods 202A general method 202Choosing the correct dilution of antibody 203Fluorescent labels 204Peroxidase 204Alkaline phosphatase 207Glucose oxidase 209Galactosidase 209Immunogold 209Some general procedures 210

7. Controls and problem solving 213Controls 213Problem solving 213

8. Detecting two antigens on the same section 214

9. Cytological preparations 215

10. Quantification 218

11. Equipment 218

References 219

6. Calcium and pH imaging in living cellsRichard M. Parton and Nick D. Read

1. Introduction 221

xii

Contents

2. Study of calcium and pH in living cells 221

3. Imaging intracellular free calcium and pH 222

4. Fluorescent dyes for free calcium and pH 223Properties of calcium and pH dyes 223Intracellular dye behaviour 225Single wavelength dyes, ratiometric dyes, and ratiometric dye-pairs 230

5. Introducing calcium and pH dyes into living cells 231General considerations 231Ester loading 232Low pH loading 235Scrape loading 235Electroporation 235lonophoretic microinjection 236Pressure microinjection 236Quantifying the extent of dye loading 237

6. Equipment for fluorescence microscopy 237Fluorescence microscopes 237Objectives 238Dye excitation sources for ion imaging 239Filters for ion imaging 239

7. Fluorescence imaging systems 240General requirements 240Conventional fluorescence imaging 241Confocal imaging 243Multiphoton imaging 244Imaging with multiple detectors 245

8. Optimizing the performance of imaging systems 245

9. Handling experimental material on the microscope stage 248

10. Digital image processing 249

11. Ratioing 249

12. Quantitative ion imaging 252Image quality and quantitative imaging 252Signal-to-noise ratio 254Numerical data extraction 256Quantitative ratio imaging 257Calibration of dye response 260Statistical analysis of image data 264

13. Visual presentation of image data 267Visual image enhancement 267Preparation of digitized figures and plates for publication 267

14. Combining ion imaging with other experimental techniques 269

15. Critical controls for intracellular ion imaging 270

xiii

Contents

Acknowledgements 271References 271

7. Reflection-contrast microscopy 275J. S. Ploem, F. A. Prins, and I. Cornelese-ten Velde

1. Introduction 275Methodology 275Applications 275Review articles 276

2. Optical systems for RCM 276Early developments in reflected-light microscopy 276New developments in reflection-contrast microscopy 277Modern reflection-contrast microscopes 280

3. Image formation 286General 286Image formation in RCM of living unstained cells 289Image formation in RCM of stained specimens 289

4. Applications 290General 290Special applications 295

5. Specimen preparation 298General 298Immunohistochemistry 298Fixation and embedding 301Sectioning 303Immuno- and histochemical staining for RCM 305Mounting and examining sections on microscope slides 307

6. Summary 308Acknowledgements 309

References 309

8. Histomorphometry 311A. J. Reynolds

\. Introduction 311

2. Microscopy 313Specimen preparation 313Obtaining an image 318Calibration 319

3. Linear measurements 321Intercept measurement 321Point counting 324

xiv

Contents

4. Automated measurement 327Measurement with an interactive computer system (digitizer tablet) 327Semi-automatic image analysers 329

5. Histomorphometry 337


Instrumentation and sources of supply 338

References 339

9. Near-field optical microscopy 341Niek F. van Hulst

\. Introduction 341Optical microscopy 341Probe microscopy 342Breaking the diffraction limit 342Scope of this chapter 344

2. Instrumentation 344Probes and distance regulation 344Antenna-type or 'appertureless' near-field scanning optical

microscopy 346Aperture-type near-field scanning optical microscopy 346Photon scanning tunnelling microscopy (PSTM) 349Distance regulation: shear force microscopy 349Conclusion 350

3. Applications 355Single fluorophores and proteins 355Monolayers and aggregates 358Virus 362Cellular surface and cytoskeleton 362Chromosomes and fluorescence in situ hybridization 363

4. Conclusions 367

5. Future outlook 369


References 370

10. Introduction of materials into living cells 3731. Particle bombardment as a means of DNA transfer into

plant cells 373

Christian Schdpke and Claude M. FauquetIntroduction 373Practical considerations 374Identification of cells transformed with reporter genes 380

xv

Contents

2. Microinjection as a preparative technique for microscopicalanalysis 386

H. F. PatersonAdvantages of microinjection by glass capillary needle 386Equipment required for microinjection of adherent mammalian

cells 387Preparation of materials for microinjection 391Microinjection technique 392Microscopical analysis of microinjected cells 394


References 396

11. Surface fluorescence microscopy withevanescent illumination 399D. Axelrod

1. Fluorescence at surfaces 399TIRF for biochemical samples 399TIRF for biological samples 401

2. Theory of TIRF 401Single interfaces: intensity and polarization 401Intermediate dielectric layers 405Intermediate metal film 406

3. Optical configurations 407Inverted microscope TIR with prism on top 407Inverted microscope TIR with prism below 410Upright microscope TIR with prism below 411Inverted microscope TIR without a prism 414Rapid chopping between TIR and epi-illumination 417

4. General experimental suggestions 417

5. Applications of TIRF microscopy 420

6. Comparison with other optical sectioning microscopies 422


References 423

12. Nanovid microscopy 425Greta M. Lee

1. Introduction 425Use of nanovid microscopy to analyse molecular mobility in

membranes 428Use of nanovid microscopy to study the extracellular matrix 428

xvi

Contents

2. General information on equipment and methods 432Microscope 432Camera 433Image processor and frame grabber 433Image storage 434

3. Image analysis to analyse molecular mobility (singleparticle tracking) 434

References 435

Appendix I 437

Appendix II 443

Index 447

XVll

Contributors

D. AXELRODUniversity of Michigan, Biophysics Research Division, 930 North University,Ann Arbor, Michigan 48109-1055, USA.

I. CORNELESE-TEN VELDEDepartment of Pathology, Leiden University, PO Box 9600, 2300 RC Leiden,The Netherlands.

CLAUDE M. FAUQUETILTAB/ORSTOM, The Scripps Research Institute, Division of Plant Biology- BCC 206,10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

SUSANNE F. IMRIE34, Springfield Road, Wallington SM6 OBB, UK.

A. J. LACEYDepartment of Biology and Biochemistry, Brunei, The University of WestLondon, Uxbridge, Middlesex UB8 3PH, UK.

GRETA M. LEEThurston Arthritis Research Center, CB 7280, 5107 Thurston Building, Uni-versity of North Carolina, Chapel Hill, NC 27599, USA.

A. R. LEITCHSchool of Biological Sciences, Queen Mary and Westfield College, Universityof London, London El 4NS, UK.

WILLIMAILEInstitut fur Zelltechnologie e.V., Technologie-Park Warnemiinde, Friedrich-Barnewitz-Str. 4, D-18119 Rostock-Warnemiinde, Germany.

MICHAEL G. ORMEROD34, Wray Park Road, Reigate RH2 ODE, UK.

RICHARD M. PARTONMolecular Signalling Group, Institute of Cell and Molecular Biology, Univer-sity of Edinburgh, Rutherford Building, Edinburgh EH9 3JH, UK.

H. F. PATERSONCRC Centre for Cell and Molecular Biology, Chester Beatty Laboratories,Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK.

Contributors

J. S. PLOEMPres. Kennedylan 256, 2343 GX Oegstgeest, The Netherlands.

F. A. PRINSDepartment of Pathology, Leiden University, PO Box 9600,2300 RC Leiden,The Netherlands.

NICK D. READFungal Cell Signalling Group, Institute of Cell and Molecular Biology, Uni-versity of Edinburgh, Rutherford Building, Edinburgh EH9 3JH, UK.

A. J. REYNOLDSExperimental Techniques Centre, Brunei, The University of West London,Uxbridge, Middlesex UB8 3PH, UK.

CHRISTIAN SCHOPKEILTAB/ORSTOM, The Scripps Research Institute, Division of Plant Biology- CAL 7,10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

P. J. SHAWDepartment of Cell Biology, John Innes Centre, Colney, Norwich NR4 7UH,UK.

WALTER STEFFENMikroskopiezentrum, Institut fur Zellphysiologie und zellulare Biosysteme,Fachbereich Biologie, Universitat Rostock, Universitatsplatz 2, D-18051Rostock, Germany.

A. T. SUMNER7 Smileyknowes Court, North Berwick, East Lothian EH39 4RG, UK.

NIEK F. VAN HULSTApplied Optics Group, Faculty of Applied Physics, MESA Research Insti-tute, University of Twente, PO Box 217, 7500 AE Enschede, The Nether-lands.

DIETER G. WEISSLehrstuhl fiir Tierphysiologie, Institut fur Zellphysiologie und zellulareBiosysteme, Fachbereich Biologie, Universitat Rostock, Universitatsplatz 2,D-18051 Rostock, Germany.

ROBERT A. WICKPricewaterhouseCoopers LLP, Ten Almaden Blvd., Suite 1600, San Jose, CA95113, USA.

xx

Abbreviations

Ab antibodyAFM atomic force microscopyAM acetoxymethylAOM accousto-optical modulatorb.f.p. back focal planeBSA bovine serum albuminCaMV cauliflower mosaic virusCCD charge coupled deviceCHO Chinese hamster ovaryCLSM confocal laser scanning microscopeDAB diaminobenzidineDAPI 4' ,6-diamidino-2-phenylindoleDIC differential interference contrastDNP dinitrophenolDPX distrene, dibutylphthalate, xylolDTT dithiothreitolEM electron microscopyFAC fluorescent analogue cytochemistryFISH fluorescence in situ hybridizationFITC fluorescein isothiocyanateFVN field of view numberFWHM full width half-maximumGFP green fluorescent proteingfp gene encoding green fluorescent proteinGUS (3-glucuronidaseHRP horseradish peroxidaseIAD illuminating aperture diaphragmIFD illuminated field diaphragmIg immunoglobulinIGSS immunogold silver stainingILTAB International Laboratory for Tropical Agricultural BiologyLM light microscopyLUC luciferaseluc gene encoding luciferaseLUT look-up tableNA numerical apertureNIH National Institutes of HealthNOR nucleolar organizing regionsNP-40 Nonidet P-40NSOM near-field scanning optical microscopy

Abbreviations

PA 'pro analyse' quality reagentsPBS phosphate-buffered salinePCR polymerase chain reactionPHA phytohaemagglutininPMSF phenylmethylsulfonyl fluoridePO peroxidasePOL polarizationp.s.f. point spread functionPSTM photon scanning tunnelling microscopyRCM reflection-contrast microscopyRGB red, green, blueROI region of interestRSE relative standard errorSCV settled cell volumeSDS sodium dodecyl sulfateSIT silicon intensifier targetS/N signal-to-noiseTEM transmission electron microscopyTIFF tagged image file formatTIRF total internal reflection fluorescenceTPCLSM two-photon laser scanning microscopeuidA gene encoding p-glucuronidaseVEC video-enhanced contrastVIM video-intensified microscopyX-gluc 5-bromo-4-chloro-3-indolyl-p-D-glucuronide cyclohexyl-

ammonium salt

xxn

Basic optical microscopyA. J. LACEY

1. IntroductionThe perception of, or the sight of, an object whether in a microscope or in aneveryday situation is the result of a complex process. It begins with light inter-acting with the object. The modified light is collected by the lens of the eyeand brought to a focus on the retina of the eye. The nerve responses in theretina are interpreted by the eye/brain system programmed to a greater orlesser extent by previous experience.

The area of the specimen seen by the eye is the perception of the extensionin x and y directions, while the experience of the of the z direction is the per-ception of depth.

2. The microscope and its useProtocol 1 gives a practical guide to establishing the potential of the hardwareof a microscope.

Protocol 1. Examination of the microscope potential

EquipmentMicroscope and accessories

A. Lamp(s)

1. Trace the power supply from the mains to the light source. Discovertype of lamp available, i.e. tungsten halogen, high pressure mercury,or other. Locate centring screws for lamp relative to its collector lens.There may be two separate light sources one for transmitted andanother for epi-illumination or reflected light work. Locate the sliderwhich blanks off the incident light train. There may be a focus controlfor the lamp collector lens. Establish its function (see Kohler illumina-tion protocol. Protocol 2).

1

A. J. Lacey

Protocol 1. Continued

2. Find voltage control for tungsten light and the colour temperaturesetting.

3. There may be neutral density filters in the light path.

B. Condensers

1. Note carrier of condenser and find centration screws.

2. Note type, i.e. universal, bright-field with top lens control by screw offor flip top.

3. Note the appearance of the lamp side of the condenser, i.e. the phaseannuli, dark-field stop, or Wollaston prism, illuminating aperture dia-phragm, and any centration facility for each of these. Engravings mayinclude lens corrections such as NA (numerical aperture), apochro-matic. Direction of shear is shown in DIC condensers.

C. Objectives (usually a range of objectives in a rotatable turret)

1. Numerical aperture (NA), e.g. 0.65 or 1.3 (possible iris for control ofNA).

2. Magnification 40:1 (or just 40), 100, X100 (infinity corrected) x 40(angular magnification) x 100.

3. Tube length: 160 mm or infinity (°°).

4. Coverslip (thickness) 0.17 (correction collar for high dry NA).

5. Immersion (otherwise dry): usually oil but sometimes water or glycerine.

6. Lens type: Plan, Phaco, Apo, Fl, etc.

7. Manufacturer and code number.

D. Eyepieces

Often inserted as a pair in a head with interpupillary distance control. Theright-hand one may have a diopter control for balancing the focus of thetwo eyes. The head may have a slider to bring in a beam splitting prism totake all or some of the light to a camera port.

1. Engravings:

(a) Magnification: x 10, x 15.

(b) Field of view number (FVN): 18, 14. This gives an indication of thefield of view but needs converting to mm by reference to objectivemag. and tube factor—see note (a).

(c) Type (field): wide-field (WF).

(d) Corrections: compensating.

(e) High eyepoint: spectacles symbol.

1: Basic optical microscopy

2. Note whether there is a magnification changer present in the tube ofthe microscope and whether such a changer has a telescope (phase)position and a focus control for it.

Notes:(a) The field of view in mm =

The tube factor may be X 1.25 for a binocular head but it can be found bycalculation substituting a measured field of view in the above equation.

(b) It is important to match the lenses in use at any one time. Thus the veryhighest corrected objectives should be used with highly corrected con-densers and appropriate eyepieces.

3. Summary of the process of image formation in themicroscope

The summary is illustrated graphically in Figure 1.

(a) The specimen is illuminated.(b) The specimen and the light interact.(c) Part of the light is scattered.(d) Part of the light is unchanged.(e) The objective collects the light.(f) What is collected by the objective is seen (as an optical transform of the

specimen) in the back focal plane (b.f.p.) and passes on through it.(g) The light issuing from the b.f.p. passes to the primary image plane where

it forms an interference pattern (a further optical transform).(h) This interference pattern is the primary image produced by the objective

and is a magnified replica of the specimen (see Figure la).(i) The light passing on is further modified by angular alteration in the eye-

piece (Figure 1b).(j) It passes out through the exit pupil of the microscope and into the

entrance pupil of the eye with an angle which is perceived as furthermagnification (Figure 1b).

3.1 Lamp collector lens and condensersThe light issuing from the filament is collected by a lamp collector lens whichfocuses it on to the illuminating aperture diaphragm. The condenser then col-

Figure 1. (a) Summary of image formation in the microscope. Main steps in the processare indicated on the right. P, Q, R represent features in the specimen which are imaged atP1, Q1and R1. (b) Geometrical optics of the light microscope. fe = focal length of the eye-piece; fo = focal length of the objective; To = tube length (notionally = 160 mm); h =specimen dimension; H = intermediate (primary) image dimension; H' = final (virtual)image dimension; Dv = least distance of distinct vision (250 mm).

lects the light and puts it as a cone whose apex falls into the specimen. Thispractical process is given in Protocol 2 (Kohler illumination). Condensers areprecision lenses with colour and spherical aberration corrections and areoften capable of several contrast techniques—see under contrast methods(Section 8).

3.1.1 Light matter interactionsThe light then interacts with the specimen in some or all of the following ways:

absorptiontransmissionreflectionrefractiondiffractionpolarizationabsorption and subsequent re-emission—fluorescence or phosphorescence

The cone of light which entered the specimen is scattered to a greater orlesser extent by the specimen (Figure la) and some may be lost out of thesytem. For the simple case the central ray of light passes on up the optical axis


A. J. Lacey

of the microscope. Some of its energy may be scattered at an angle by diffrac-tion or refraction from this straight line. The angle, of diffraction for example,will be related to the fineness of the patterns in the specimen. Protocol 3 illus-trates this process.

3.1.2 ObjectivesThe objective lens in a microscope collects the undeviated light and the scat-tered light to an extent determined by the angular aperture of the objective ormore precisely by its numerical aperture (NA).

That light which is collected by virtue of the numerical aperture passes onthrough the back focal plane of the objective (b.f.p.) and is brought to a plane(the primary image plane) where the various components of the light (thedirect light and the scattered light) interfere with each other and form aninterference pattern called the primary image. This image is a magnifiedreplica of the specimen. The NA of the objective is an important componentin determining the resolving power of the microscope as shown in Equation 1.

where lambda is the wavelength of light used (e.g. 500 nm or 0.5 jxm for greenlight) and NAobj the numerical aperture of the objective. The resolving powerof the objective can be calculated thus for a 0.65 NA objective as about500 nm or 0.5 |xm and for a NA of 1.3 as half these values (twice the resolvingpower) (see Protocol 3 for the explanation in practical terms).

One of the engravings on the objectives indicates the primary magnificationthat is the ratio of the primary image size to that of the specimen. The primaryimage resulting from the objective is then effectively further magnified by theeyepiece lenses, and seen by the eye.

3.1.3 EyepiecesThe light passes on from the primary image plane and is altered in angularterms as it passes through the eyepieces. This alteration is perceived by theeye/brain as the final magnification of the microscope. The eye(s) is placedwith its entrance pupil at the exit pupil or Ramsden disc of the microscope.The Ramsden disc can be found by following Protocol 2.

The eyes should be set comfortably over the eyepieces—both open even ifthere is only one eyepiece. They will eventually learn to be in a relaxed state,i.e. focused on infinity and viewing the image as though it were at infinity. Thedistance apart of the binocular eyepieces (the interpupillary distance) shouldbe set by finding the position of a single image seen through both eyes. Theseparation given on the scale should be memorized. The left eye system isfocused first by the microscope focus control, shielding the right eye by apiece of paper rather than closing it, and then the right eyepiece is brought toa comfortable focus, shielding the left eye, by altering the diopter setting onone of the eyepieces. Again it can be noted for future reference.


4. Kohler illuminationIn order to obtain an even distribution of light on the specimen and an evenbackground intensity a sequence of lamp collector and of condenser lenses isused.

The principles of the method are:

(a) A lens in front of the light source places an image of that light source at aposition which is not in the plane which contains the specimen.

(b) A second lens (the condenser) puts an image of the surface of the firstlens onto the specimen to be examined and does so with as wide an aper-ture as possible to illuminate the specimen.

Protocol 2. To obtain Kohler illumination in a transmitted lightmicroscope and to establish conjugate planes

EquipmentMicroscope with light source, illuminated Contrasty specimen such as a periodic acidfield diaphragm (IFD), illuminating aperture Schiff/Light Green or Masson trichromediaphragm (IAD) (an optional extra would stained specimenbe a phase telescope)

Method1. Examine the microscope (for an unfamiliar machine) carefully to

establish the physical control procedures. Trace the pathway from themains wall switch through to the tungsten or quartz halogen lightsource with its intensity control filters, IFD, condenser with its IAD,and focus control, objectives (type and range) to the eyepieces (againtype and range). There may be supplementary magnification changersin the tube of the microscope and other modifications may includelight splitting prism in the trinocular head.

2. The microscope may also be capable of reflected light microscopywith filter blocks appropriate for fluorescence work. These should beslid out of the light train for transmitted work.

3. Switch on the light and check its centration, following the maker'sinstructions, with respect to the lamp collector lens.

4. Using the contrasty specimen focus an image of it through a lowpower objective such as x 10.

5. Looking through the eyepiece partially close the IFD until it is seen inthe field of view, i.e. in the plane of the specimen.

6. Centre the image of the IFD by the use of centration screws on thecondenser. Focus the condenser to give the field iris a sharp image. Inmoving the condenser focus you may notice that the image of the IFDhas a coloured edge. This should be of one colour only and change

A. J. Lacey

Protocol 2. Continuedevenly otherwise suspect poor alignment. Check alignment instruc-tions as appropriate.

7. Open the IFD until it is just outside the field of view of the microscope.This procedure will also provide more critical centration. It is at thisstage that in really low power objectives the image of the field irismay not fill the field of view. In this case either a supplementary lensmay be brought into the light path below the condenser or the flip toplens removed from the condenser. There may then be need to refocusthe condenser (step 6).

8. Take out the eyepiece and insert the phase telescope (or use the built-in Bertrand lens), to focus the telescope on the back focal plane(b.f.p.) of the objective. This can be recognized by seeing the image ofthe IAD in view. Test that this is the case by opening and closing thatdiaphragm. There may be capacity for centring the aperture imagerelative to the b.f.p. If this is so then do so but being careful not tomeddle with the centring screws of the condenser itself which youhave already been set. Adjust the IAD so that it fills about 70% of theb.f.p. of the objective. You will notice that some glare disappearsfrom the side of the body tube of the microscope as you make thisadjustment. You may also notice that there is an image of the lightsource in the centre of this bright illuminating aperture. This is as itshould be. If there is a facility for centring the lamp filament then doso with respect to the image of the condenser iris and the b.f.p. Thesethree are said to be conjugate (see later). The presence of a diffusernear the light source may not allow the image of the filament to beseen so the maximum brightness point should be centred.

9. Replace the eyepiece and assume that this is the best situation for theparticular combination of condenser and objective settings. Excessivebrightness in the image should be reduced by the insertion of neutraldensity filters into the light path and not by reducing the IAD. Forvisual microscopy it is permissible to reduce the intensity of the lightsource but this will increase the warmth of the colours and is notappropriate for colour photomicrography (see later).

10. For a change in objective, i.e. to a x 40, check steps 5-9. You willprobably find that all that is required is to increase the IAD to matchthe increased size of the b.f.p. in the higher numerical aperture (NA)objective. When returning to a lower NA objective remember to againcheck steps 5-9.

11. To complete the understanding of the conjugate planes in the micro-scope place a piece of ground glass or lens tissue at right angles tothe light path just above the eyepiece. Move the tissue or glass alongthe light path towards or away from the surface of the eyepiece. You

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will discover a place where the apparent disc of light is at a minimumdimension. This disc of light is the exit pupil of the microscope, other-wise known as the Ramsden disc. In correct conditions it also acts asthe entrance pupil of the eye. It is about 3 mm in diameter and is con-jugate with the b.f.p. Note for spectacle wearers it is some greater dis-tance (15 mm) from the eyepiece and an appropriate eyepiece will bemarked with a engraving of spectacles.

12. Compile a list of the conjugate planes in which that of the b.f.p. isincluded. Note that this series is as follows: lamp filament, IAD,b.f.p.of objective and Ramsden disc.

13. Find the other series which includes the IFD, the specimen, the primaryimage, and the retina of the eye (or the emulsion of the film or chip ofthe video camera). The primary image lies in the eyepiece just out-side the inner focal point of the eyepiece eye lens and a diaphragm(the field diaphragm of the eyepiece) there gives the image a clearedge. A field lens also occurs in the eyepiece which controls the areaof the primary image entering the eye.

14. Kohler illumination successfully keeps the irregularities of intensity inthe light source out of the series which contains the specimen.

These principles are the basis for Protocol 2. The planes which contain thelamp filament or its image comprise one series and those containing the speci-men and its image comprise another and are set out in diagrammatic form inFigure 2 and in Table 1.

Figure 2. A ray diagram of Kohler illumination showing the positions of the two sets ofconjugate planes. Solid lines represent rays of light moving from left to right arising fromthe centre of the lamp filament to the near margin of the lens. Dotted line is a ray passingthrough the centre of the lamp collector lens. Vertical solid lines with arrow heads arelenses, while vertical solid lines without arrow heads are diaphragms (irises). Verticaldotted lines are planes in which specimen, back focal plane (b.f.p) of objective and imageare situated. Zigzag represent the lamp filament and its subsequent images.

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Table 1. Conjugate planes in Kohler illumination

Series A (aperture set) Series B (field set)

Lamp filament (source) Illuminated field diaphragm (IFD)Illuminating aperture diaphragm (IAD) SpecimenBack focal plane of objective (b.f.p.) Primary imageRamsden disc Retina of eye

All modern microscopy should be commenced by setting-up Kohler illumi-nation. The capacity of your microscope to set up Kohler illumination isthereby tested. One of the situations often met with is that the lamp collectorlens may have a diffuser surface. This is sometimes the surface next to thelamp and it is so treated to break up the image of the irregularities in the lightsource. Other variations may be in having a ground glass just near the field irisfor the same reason. The condenser aperture iris is sometimes at a consider-able distance from the front focal plane of the condenser making for difficultyin losing its image from the series conjugate with the specimen.

5. Resolution in the microscopeIt was stated in Section 3.1.2 above that the NA of the objective is paramountin determining the details in the image. Protocol 3 sets out an example of howto demonstrate this. By combining the magnifying power of the objectives andeyepieces to produce an equal magnification it is readily possible to show thatthe NA of the objective is the key factor (see Figures 3a-c).

Protocol 3a. To demonstrate empty magnification

EquipmentMicroscope with objectives x 40, NA 0.65, The diatom Navicula lyra mounted in DPXand x 10, NA 0. 25, and eyepieces x 25 and or other resin8

X 6

Method

1. Observe the diatom with the x 40, NA 0.65 objective together with a x 6eyepiece to give a total magnification of X 240. Observe the detailpresent in the image as the objective is capable of resolving spacing ofabout 500 nm.

2. Repeat with an objective x 10, NA 0.25, and using the x 25 eyepiece.The combined objective/eyepiece will give a magnification of x 250.Note the general outline of the diatom is the same but in the image ofits surface there is no detail.

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3. Infer that there is excess magnification in the second case. The NA0.25 objective is only capable of resolving spacings of larger than 1200nm and the pattern in the diatom is smaller. Therefore there is surplusor empty magnification present.

4. The regular pattern of dots in the frustule of the diatom can be seen ifthe NA of the objective is greater than 0.3. The pattern is not resolvedin the case of the lower NA objective.

aA test plate can be obtained from Micro Instruments Ltd. in which five diatoms are mountedincluding N. lyra. Recommended combinations of NA 0.3 and total magnification (x 100)required to see the detail are engraved on the divisions of the test plate. Unless there is moredetail revealed by additional magnification then the addition is described as empty magni-fication (see Figure 3). Some additional magnification however makes for more comfortableviewing.

Protocol 3b. Pleurosigma resolved

EquipmentOil immersion objective with numerical Prepared slide of Pleurosigma angulatumaperture 1.3, and with a iris in the b.f.p.capable of restricting the b.f.p. to NA 0.8

Method

1. Set up Kohler illumination.

2. Find and centre the diatom using the objective with its iris open to full1.3.

3. Close down the condenser iris to as near a pin-hole as possible. Noteimage of the specimen contains a hexagonal pattern in the diatomsurface (see Figure 3c).

4. Remove eyepiece and observe b.f.p. of objective. Note presence ofbright central image of the aperture iris (conjugate) and also the sixcoloured images of this iris around the margin of the b.f.p. These arethe six first order diffraction images surrounding the zero order light(Figure 3a).

5. Close the objective iris noting as you do so that you are occluding thefirst order diffraction (Figure 3b), i.e. preventing it from leaving theb.f.p.

6. Replace the eyepiece and observe the loss of the hexagonal pattern inthe surface of the diatom (Figure 3d). Reopen the iris and retrieve thepattern.

7. Repeat steps 1-6, noting appearance of images and of the alteredb.f.p.

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Protocol 3b. Continued

8. Conclude that the pattern is only present in the image when the firstorder diffracted light contributes to the formation of the image. Whenthis occurs the microscope is said to have resolved the pattern. (NoteProtocol 6 gives a practical illustration of the importance of the zeroorder light.)

Although Equation 1 gives the basic resolution equation of the microscope

it is better to think of the effective NA as being the mean of the NAs of theobjective and the condenser. It was mentioned on page 4 that the condenser

throws an oblique cone of light on to the specimen. In normal Kohler the

Figure 3, The relationship of the image to what light passes through the back focal plane(b.f.p.) of the objective. The objective is a Zeiss Planapo NA 1.3-0.8, x 100, oil immersionlens. The specimen is the diatom Plourosigma angulatum [scale bar = 20 |j.m). (a) Theb.f.p of the objective with fully opened iris allowing the central zero order light and the(six) first order light to pass through to forming the image with detail resolved in (c). In(b) the iris in the objective is (almost) occluding the first order diffracted light and produc-ing an image in (d) which fails to resolve the detail in the diatom.

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illuminating aperture is about 70% of the NA of the objective. Equation 2 is abetter version of the resolution formula:

provided NAobj is larger than NAcond.Protocol 4 sets out a technique for making make a test specimen of point

sources of light, the testing of the performance of objectives, and the practicalillustration of the Rayleigh criterion. For a definition of the Rayleigh criterionsee Chapter 3, Figure 4, page 82.

Protocol 4. To prepare and use a star test object

Equipment and reagentsClean coverslips (No. 1 or 0.16 mm thick) Vacuum coaterand slide DPX or other mounting medium

Method

1. Holding a coverslip by the edges, place it horizontally in a sputtercoater.

2. Coat one surface with an appropriate metal alloy until opaque.

3. Blow on the metallized surface of the coverslip to remove any dustparticles.

4. Cement the coated side downwards on to a microscope slide.

5. Examine through microscope for the tiniest of holes.

6. Having found the tiniest of holes increase the eyepiece magnificationas far as you are able, until the rings of light are visible around thecentral disc of light.

7. Observe and note carefully the appearance of these Airy disc patternswhich are the images of the point sources of light. Use a range ofobjectives and make careful notes of the appearance above andbelow focus.

8. Examine the range of Airy disc patterns over the whole field of viewlooking for distortions at the edges of the field of view.

9. Note again their appearance when viewed in green, blue, or red light.

10. Returning again to white light endeavour to find two holes which areclose or touching each other. There will be some holes which lookdumb-bell-shaped. Are these two overlapping images or are they oneof a single oddly shaped hole?

Visual acuity and patient observation with accurate note taking make thisspecimen a very testing one not only for the student of microscopy but also for

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a biologist who is anxious to know the quality of the performance of his/herobjectives and microscope system.

In the present context it is only to demonstrate that an image of a pointsource of light is a diffraction pattern. The radius (d') of the first dark ring isproportional to the wavelength of the light in use (smaller for blue and largerfor red) and inversely proportional to the numerical aperture of the lens usedto produce it. The formula is given as d' = 0.61 lambda/NA (Equation 1).

That figure illustrates the intensity scan of the images of two point sourcesof light close together. They are sufficiently close for the diffraction rings (firstand second order) of one to coincide with the main peak (zero order) of theother. The sum of the zero order peak and the other orders of diffracted lightis given by the dotted line. If the difference between the peaks and the centraltrough of the dotted line is perceptable by the eye (usually taken as 16%) thenthe two points are resolved as two points (Rayleigh criterion of resolution).Video detection is more sensitive than that of the eye in that it is capable ofdifferentiating less than 16% differences. The Sparrow criterion of resolutionis where the dip in intensity between the Airy disc overlapping patterns ismade visible through the aid of video-enhanced detection. Space does not allowa fuller treatment of the subject here. Point sources for fluorescence micro-scopy can be made by utilizing the 0.1 (xm or smaller fluorescent P. S. Speckbeads available from Molecular Probes Inc. See Chapter 3 for further informa-tion on the point spread function. The Airy disc pattern obtained may showaberrations due to poor setting-up rather than intrinsic faults in the lenssystems so beginners must be cautious in rejecting lenses.

6. MagnificationMagnification is a two stage process in the microscope. The first stage is wherethe objective produces a real (inverted) magnified image (the primary image).The second stage when the rays emerging from the b.f.p. of the objective passthrough the eyepiece lenses to be thereby altered in angle and to present theprimary image to the eye as still further magnified. Total magnification shouldbe at least enough to bring the detail resolved by the microscope to the resolv-ing power of the eyes, i.e. about 2 minutes of arc. Magnification should also bewithin the limits set by the working pupil of the eye, i.e. within the limits of170 X NAobj to 1000 X NAobj. Figure 4 illustrates the examination of tissuesections with various combinations of NA in the objective and magnifications.

7. Interim summarySummary of image formation in the microscope so far:

(a) The image in a microscope is a replica of the specimen constructed byinterference of light waves emanating from the specimen.

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Figure 4. Magnification and resolution. Three photomicrographs taken through objec-tives of different apertures and magnifications, all printed at a magnification of 400:1. (a)Objective 4/0.16. Final magnification 2500 x MA, The image is unsharp and obviouslyover-magnified, (b) Objective 10/0.25. Final magnification 1600 x NA. The image still lacksinformation in fine details, (c) Objective 25/0.65. Final magnification 615 x NA, In thisimage the fine detail is sharply rendered. Scale bar = 50 ^m. From ref. 1 with permission.

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A. J. Lacey

(b) The microscope is, or is not, capable of resolving the detail in the speci-men, when illuminated by a particular wavelength of light, by virtue ofthe numerical aperture of the objective.

(c) The resolution is made available to the eye by the combined magnifyingpower of the objective and the eyepieces.

(d) Resolution however may not be visible unless contrast is present.

8. Contrast methodsThe perception of the detail in the image is only possible if there is sufficientcontrast presented to the eyes between the background and the specimen andbetween the various features in the specimen. The normal viewing in a trans-mitted light microscope is by presenting the specimen on a bright background.If however the background is too bright nothing will be seen. The contrast isproportional to the difference in intensity of the background and the specimenand inversely proportional to the intensity of the backgound.

A series of practical protocols is now given to illustrate the achievement ofcontrast in the image.

8.1 Bright-fieldBright-field (Protocol 5) contrast is due to the kind of optical system used, i.e.the specimen is seen against a bright background and its contrast is largely theresult of absorption by the stains in the specimen with its required chemicalfixation and mounting procedures of that specimen or by the scattering powerof the specimen. The numerical aperture of the objective also determines thedepth of the material which is in-focus at any one plane of focus at any onetime. That is information about the z direction in the specimen likely to beconfused by out-of-focus light coming from planes above and below the planeof focus (see later chapters for further treatment of this situation). Sectioningof a large specimen is required to produce as near a single layer in the z direc-tion as possible. However the use of reduced illuminating aperture diaphragm(IAD) in achieving contrast in weakly stained material is reviewed as are theuses of colour filters in stained specimens.

Protocol 5. Bright-field microscopy; contrast assessment

Equipment and reagentsMicroscope with Kohler illumination Set of colour filtersCheek cells mounted in saliva or onion Masson trichome and/or periodic acidscale inner epidermis mounted in water Schiff stained animal sections

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Method1. Set up Kohler illumination as in Protocol 2.2. High contrast situation: examine the stained specimen in white light.

Recall that the contrast seen here is by differential absorption/transmission of the many wavelengths comprising white light. The redportions transmit red light and the green areas are those transmittinggreen light of about 500 nm wavelength.

3. Observe the effects of using monochromatic light obtained by placingfilters in the light path. Use filters of complementary colour to bringabout increased contrast for example a green filter will darken a redstained area of the specimen (see Figure 5). Filters of the same colourcan reduce the impact of stains and make excessively over-stainedareas more transparent.

4. Low contrast situation: examine the cheek cells or the onion cells inKohler illumination in white light as for step 1. Note virtually no con-trast, i.e. nothing visible. There may be some visibility in the onionpreparation by virtue of the absorbence of light in the vertical walls.

5. Try closing down the IAD and note the improvement in contrast. Thenuclei and the cytoplasm of the cheek cells and also that of the onioncells will become visible. Note however that this is strictly speakingnot an appropriate technique as a good deal of the information in theimage is 'rotten', i.e. has diffraction rings round small features.

Conclude from this protocol that staining thin sections is a powerfulmethod of obtaining contrast between the specimen and the backgound andbetween features in the specimen. Often however this staining procedureleads to loss of lifelike dimensions or produces chemical artefacts. For livingcells refer to Protocol 3 for phase-contrast, and Protocol 4 for differentialinterference contrast (DIC).

8.2 Dark-fieldThe theoretical background of the contrast techniques is avoided but the caseof high power dark ground (Protocol 6) is used to illustrate, in the image aswell as the change of contrast, the importance of the zero order light in imageformation. Its occlusion in the b.f.p. can be shown in this case to producereversed contrast in Figure 6.

For low power objectives it is sometimes possible to use the illuminatingannulus in the phase condenser appropriate for a higher objective NA condi-tion. The annulus then acts as a kind of simple patch stop.

Note: Rheinberg illumination. This is done exactly as for dark ground sim-ple patch stop but substituting complementary colours for the central stop andthe surrounding illumination. A pleasing image of the specimens in the colourof the surrounding annulus on a background the colour of the central stop.

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Figure 5. The effect of colour filters on contrast. The specimen (frog stomach section) isstained with periodic acid Schiff and Light Green, photographed on Kodak Technical Panfilm, and developed for medium contrast. The appearance of the micrographs is closelysimilar to that seen directly with the eye. (at No filter. The tips of the cells, densely stainedmagenta, appear slightly darker than the rest of the specimen which is in shades ofgreen, (b) Red filter. The magenta areas now appear colourless and the green stainednuclei are dark and more conspicuous, (c) Green filter. The green stained nuclei cannotnow be seen, while the magenta stained regions are shown in strong contrast. Scale bar= 50 ^m. From ref. 1 with permission.

Figure 6. Signifance of the zero order light in image formation: dark-field image of P.angulatum. Objective and specimen as in Figure 3. Condenser this time illuminating witha wide diameter annulus instead of a central disc, (a) The b.f.p. of the objective with its irisfully open to NA 1.3 accepts and allows through the zero order light together with the (six)first order diffracted light (rings intersecting in the centre to pass through, (c) The image isa bright-field image. (b)The iris in the objective occludes the zero order preventing it fromcontributing to image formation, (d) The image is seen to be one of reversed contrast.

Protocol 6a. To achieve contrast by the use of dark groundillumination: low power and high power systems

EquipmentTransmitted light microscope Simple patch stop

Method

1. Set up Kohler illumination and observe a slide on which there are anumber of discretely separated features such as a diatoms or in aplankton preparation.

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1: Basic optional microscope

A. J. Lacey

Protocol 6a. Continued2. Recalling that you adjusted the aperture iris to give about 66% illumin-

ation of the b.f.p. of the objective, examine the physical condenser irisand note the size of the opening.

3. Prepare a disc of transparent material of the size of the filter housingof the condenser. On to the centre of this disc place a disc of opaquematerial of the same size or slightly larger than the physical opening ofthe aperture diaphragm.

4. Place centrally in the light path as near the IAD as possible.

5. Re-examining the specimen you will probably discover that nearlyeverything is black! Adjust the focus of your condenser slightlyupwards and open the IAD. This should enable the diffracting featuresof the specimen to become brightly visible on a background of mini-mum intensity.

Protocol 6b. Method for high power dark ground

EquipmentUniversal condenser or specialist dark Immersion oilground condenser Objective with a variable diaphragm in itsSpecimen such as Pleurosigma angulatum b.f.p. (usually the NA is given as variable inas illustrated in Figure 6 this type of objective)

Method

1. Set the specimen as for bright-field conditions and note its location byreference to stage settings.

2. Remove the specimen and then for universal condensers place a dropof immersion oil on the top lens of the condenser and also anotherdrop on to the back of the slide in a position approximating to theposition of the specimen.

3. Carefully replace the slide on the stage allowing the two drops of oilto merge from their centres outwards thereby eliminating any airbubbles.

4. Recover the location of the specimen by stage controls.

5. For the oil immersion objective now place a drop of oil over thespecimen. Bring the objective into focus if necessary observing fromthe side of the microscope when oil contact is made.

6. Having focused on the specimen which will be in bright-field con-ditions if the objective iris is open, carefully close down the iris in theobjective and the image will become one of reversed contrast.

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For dry systems there are special condensers available which can beinserted once bright-field Kohler illumination has been obtained. No oil isused on the condenser in these cases.

Figure 7. Diffraction in the phase-contrast microscope, (a) Specimen of grating (phase)with line spacing about 10 |j.m. (b) b.f.p. of the phase objective NA 0.25 showing zeroorder light (0) superimposed on the darker phase plate and the other orders of diffractedlight (1 and 2) passing partly through the phase plate but mostly inside and outside of it.

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Note: careful adjustment of the settings of the iris in the objectivewill change the contrast from dark-field and back to bright-field again.It is possible by observing the b.f.p. with a telescope with these samechanges of the objective iris to see that there is a relationship of theimage contrast with the presence or absence of the zero order (illumi-nating) light. If it does not contribute to image formation then theimage is of reversed contrast on a dark dark-field (see Figure 7).

7. The oil should be carefully wiped off with lens tissue from all the sur-faces at the end of the viewing session.

A. J. Lacey

Protocol 7. To set up phase-contrast (for examining livingunstained material)

EquipmentPhase-contrast objectives and condenser Green filterPhase telescope Test specimen having a simple periodicityHigh and low contrast specimens such as in such as a diffraction gratingProtocol 2

Method

1. Set up Kbhler illumination as for Protocol 2.

2. Remove eyepiece again and insert telescope focused on the b.f.p. Notethat the phase-contrast objective has a phase plate carrying a dark ring(the phase ring) fitted in the b.f.p. The dark appearance is due to therebeing an absorbing layer in the ring to reduce the relative intensity ofthe annulus light when passing through it.

3. After replacing eyepiece, observe a specimen of cheek epithelial cells.Note the very poor contrast with normal Kohler illumination. You mayhave to reduce the illuminating diaphragm even to find them. Whenyou have done so reopen the illuminating aperture fully.

4. Swing in the appropriate sized annulus in the condenser (this may bein the slider supplied with low priced phase condensers, or in a rotat-able disc in universal condensers). The bright annulus will be of theright size if the condenser is properly focused but it may not coincidelaterally with the dark ring in the b.f.p.

5. Adjust the centration of the bright annulus to fit exactly into the phasering in the b.f.p.

6. Replace eyepiece and place green filter in the light path.

7. Note the unstained materials (or very weakly stained specimens) havegreatly improved contrast. Particularly clear are the nuclei in the cheekand onion cells. They will appear dark green (in positive phasesystems) because of their increased optical path over the cytoplasm.Granules in the cytoplasm may also be dark. There is a halo aroundeach feature. This halo is an artefact of the phase-contrast system.

8. Changing to higher objectives repeat steps 4-6. Most microscopes arecapable of holding their centration for a considerable number ofchanges of objectives and their condenser matching annuli but it isworth checking their alignment from time to time.

An experiment to illustrate the working of the phase-contrast microscope isas follows. Replace the living specimens with the diffraction grating and

22


observe the b.f.p. of the objective with the telescope. Note the appearance ofthe central ring of light (the image of the annulus) overlying the phase ring as inthe setting up procedure but there are also other rings of light as in Figure 7.These are the first order diffraction images of the annulus. Notice that theylargely pass through the b.f.p. outside or inside the phase ring. Only a smallportion of each passes through the phase ring itself.

It is important in the functioning of the phase-contrast microscope that thelight scattered by the specimen passes along a separate light path from thezero order (the central ring of light). The specimen has altered the scatteredlight by about a quarter of a wavelength (of green light) relative to the un-scattered and the phase ring puts in another quarter wave difference therebyproducing a half-wavelength in total which will give rise to interference and soan amplitude range in the image.

Note: the optical path differences (o.p.d.) are proportional to the product ofthe thickness (t) of the specimen in the z direction and the difference inrefractive index of the specimen (no) to that of the medium (nm) in which itsituated (no - nm), i.e. o.p.d. = t (no - nm). The phase-contrast image is a mapof the o.p.d. of a specimen defined by the boundaries where changes of o.p.d.produce accentuating haloes.

8.3 Phase-contrastPhase-contrast is described in Protocol 7 and the appearance of the b.f.p. ofthe objective when being used to view a grating is given in Figure 7. It is seenthat the zero order light (the bright annulus) is being reduced in intensity rel-ative to the scattered light (in this case the first order diffracted light) which ispassing outside or inside the phase plate. The zero order light is also by virtueof its passing through the phase plate being relatively changed in phase by aquarter of wavelength of green light.

8.3.1 Polarized lightCrossed polars is a very impressive technique for producing contrast in plantsections or bone material where form birefringence (due to orientatedmolecules or microfibrils) is present. Protocol 8 is a very basic illustration ofhow to determine the permitted vibration direction of an unknown piece ofPolaroid.

Protocol 8. Determination of permitted vibration direction

EquipmentPieces of Polaroid (About) 20 x 10 cm of glass 3 mm or morePreferably a polarized light microscope thickwith a rotating stage and slots (but suffi- High intensity light sourcecient for this protocol would be two pieces Section of coniferous wood unstained orof Polaroid) very weakly stained

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A. J. Lacey

Protocol 8. ContinuedA. To determine the permitted vibration direction of a piece of unknownPolaroid from first principles

Principle: when an unpolarized beam of light is reflected off a transparentsurface the reflected beam is plane polarized and its vibration direction isparallel to that of the surface of the reflecting material and at right anglesto the ray direction (see Figure 8).

1. Place the glass on a dark bench surface some distance in front of thelight source in such a way as to allow the reflection of the light beamto enter your eye (Figure 8).

2. View the light reflection through the piece of Polaroid.

3. Rotate the Polaroid slowly noting the change of intensity perceived.

4. Obtain the orientation of the Polaroid when the intensity of the beamis at a maximum.

5. Conclude from step 4 that the permitted vibration direction of thePolaroid is now parallel to the glass surface.

6. Check by observing the orientation of the Polaroid when the perceivedbeam is at a minimum intensity.

7. Conclude from step 6 that the permitted vibration direction is now atright angles to the glass surface.

8. Mark your Polaroid in one corner with an appropriate symbol.

B. To obtain contrast in an unstained or very weakly stained radial longi-tudinal section of conifer wood, between crossed polars

1. Set up Kohler illumination in the usual way.

2. Put in the polarizer usually below the condenser (in a polarized lightmicroscope it is usually on a rotatable tray marked with degrees and areference notch in the carrier). Rotate the polarizer to put 0°. This maybe effectively putting the vibration direction as determined above in aneast-west orientation.

3. With no specimen on the stage place the second piece of Polaroid(now called the analyser) in the light path above the objective, either ina purpose built slider or in a simple case as a cap over the eyepiece.

4. Rotate the analyser noting the change of intensity. At minimum intens-ity the two pieces of Polaroid are at crossed polars position. The back-ground should be as near black as possible. Note the position. Thenuncross them and insert the specimen to be examined.

5. Using the polarizer only (or having the two polarizers parallel) thespecimen can be rotated to see if there is any change of colour orintensity in its features (if there is change then suspect pleochroism).In conifer wood there is little pleochroism.

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6. Re-introduce the analyser at the crossed polars position and observethe changes of intensity pattern in the background and in the speci-men (see Figure 8). If possible rotate the specimen slowly and note thefurther changes. The black crosses in the bordered pits are known asisogyres and their presence suggest that the pits are spherulitic instructure.

7. Conclude the image is a map of the degree of birefringence (moreaccurately the optical path, i.e. the product of the thickness in the zdirection multiplied by the birefringence) and its orientation.

8. Further study of the image when the sensitive tint plate is inserted inthe slot of the purpose built microscope will provide additional colourcontrast.

The protocol goes on to show a simple crossed polars and rotation tech-nique for achieving contrast in a section of coniferous wood (Figure 8).Further use of polarized light is discussed in Chapters 3 and 11.

8.4 Differential interference contrast (DIC)DIC techniques use crossed polars and Protocol 9 sets out a technique forexamining an unstained biological specimen using a transmitted light micro-scope fitted with DIC optics. DIC is further utilized in Chapter 3, Section 2.5.6.

Protocol 9. Differential interference contrast (DIC)

EquipmentSpecimens such as cheek cells taken from Microscope fitted with plan objectivethe side of the cheek by gentle pressure lenses and carrying a set of Wollastonfrom a clean finger or wooden spatula and prisms appropriate for the objectives andmounted in saliva then covered with cover- the condenserglass are good material, or an onion skin Polarizer and analyserpreparation

Method

1. Read the instruction manual of the microscope and check thepresence of the parts required.

2. Set up Kohler illumination with the condenser set to normal bright-field conditions. You may notice the symbol on the condenser—asyou sort the parts out in the accessory box—indicating the directionof shear.

3. Insert the polarizer below the condenser.

4. Locate the cells perhaps by reference to the air bubbles in the salivaand again temporarily closing the illuminating aperture diaphragm toimprove contrast.

25

A. J. Lacey


5. Rotate in the appropriate setting (Wollaston prism) in the condenserto match the objective in use.

6. Remove the eyepiece to observe the b.f.p. of the objective.

7. Cross the polars by inserting the analyser and by tuning the upperWollaston prism to obtain the minimum intensity band in the b.f.p.Close down the aperture iris to a little larger than the dark band.

8. Replace the eyepiece and note the appearance of the image. The cellswill show a 3D quality and the direction of lighting will be apparent.This is the direction of shear referrred to in step 2.

9. Adjust the second Wollaston prism while you watch the image andnote that you can change the apparent direction of lighting and evenexperience a change in the perception of hills and valleys.

10. Infer, and it is important to do so, that the image you see is a result ofthe settings on the microscope.

11. The settings on the microscope in this way displays a contrast imageof the optical path variations present within the specimen andbetween it and the mounting medium. Each change of optical path isrepresented by an edge effect giving the image the 3D impression.

12. Colour can be obtained (in some microscopes) by referring back tostep 7 and tuning the prism to obtain a coloured band or either side ofthe zero order grey.

Note: the onion cells may show noticeable crossed polar effects in the verticalwalls which will show as birefringent brightness changing with orientation.See Chapter 3, page 120 for further information on DIC.

8.5 Fluorescence microscopyThis is a powerful contrast technique often using fluorochromes at suchlow concentrations that living biological material is unaffected by their pres-ence. Much more information and several protocols are given in Chapters 2,4,and 6.

Figure 8. Polarized light microscopy, (a) Method for determining the permitted vibrationdirection of a piece of polaroid (see Protocol 8A). (b) Appearance of an unstained radiallongitudinal section of coniferous wood (placed at 45°) viewed between crossed polars.Spherulitic structures are the bordered pits in the tracheid horizontal walls. Brightness ingeneral terms is an indication of the highly birefringent area of the specimen. Photo-micrograph on Film HP4, normal contrast development. Nikon objective x 40, NA 0.65.Bar = 100 urn. (c) Same section as in (b) but viewed with x 44 Spencer apochromaticobjective, x 6 compensating eyepiece, recorded with Sony camcorder TR805 using Hi 8SP, and video printed on a Sony Mavigraph video printer. Tracheid width = 0.40 (im.

26

1: Basic optional microscopy

27

A. J. Lacey

Figure 9. Living onion epidermal cells in different contrast techniques, (a) DIC imageshear direction NE-SW. (b) DIC shear direction NW-SE parallel to the long walls,(c) Phase-contrast of same field as (a), (d) Jamin-Lebedeff interference double focusBaker-Smith microscope with X 40 objective, (e) Dark-field transmitted light (scalebar = 100 n.m). (f) Dark-field incident light.

8.6 Provisional summary of contrast techniquesThe contrast techniques given above are largely those produced in the micro-scope techniques themselves. The use of stains and fluorescent probes areportraying maps of the chemical affinities in the specimen. Light is differen-

28


tially absorbed according to wavelength and transmitted or the energy is re-emitted at a longer wavelength. The phase-contrast and the DIC techniques aregiving contrast to changes in path (optical path) of the light passing throughthe specimen in the z direction. Polarized light is giving a map of the differentabsorption properties of the material (pleochroism) or in the case of crossedpolars technique a map of the optical path—birefringent portions, their thick-ness in the z direction, and their orientation. The removal of the backgroundintensity in dark-field microscopy and the objective lens ability to collect thelight scattered by sharp changes of refractive index in the specimen can bringabout reversed contrast in the image of that specimen.

Quantitative techniques are available for measuring optical paths by phase-contrast and by Jamin-Lebedeff interference techniques. Crossed polarsmicroscopy is very powerful in measuring refractive indices and direction ofbirefringence but biologists have not advanced as far as mineralogists in theirutilization of this technique. See Chapter 7 for reflection-contrast techniques.

Confocal microscopy (see Chapter 2) is basically a technique for preventingunwanted light from contributing to image formation. Video cameras andassociated computer software programs for image reconstruction (Chapter 3)also improve upon the basic microscopy contrast techniques by subtracting,for example, the effects of unwanted light or out-of-focus images from theimage which has been formed in the microscope. Video-intensified micro-scopy (VIM) are helpful in very low light level contexts. Chapter 3, Figure 1indicates how the conventional techniques so far described can be extendedby confocal and video microscopy in the visualization of biological materials.

8.7 Summary of contrast techniquesThe contrast in the image can be manipulated by preliminary treatment of thespecimen such as by sectioning and or staining activities which may seriouslyalter the specimen. Techniques for achieving contrast in living material arepossible by the careful control of the light passing through the back focal planeof the objective (phase-contrast) or by comparing two beams made to illumi-nate the specimen and then being recombined in interference techniques. Theimage is the result of interaction of light with the specimen. Resolving powerof the microscope is achieved by the ability of the objective to collect the dif-fracted light. A very useful exercise is to examine the same unstained specimenwith a series of contrast techniques (see Protocol 12).

9. Recording the imageThe recording of the appearance of the image can be achieved by hand-draw-ing which improves with practice! There are some drawing optical aids whichallow the image to be seen at the same time as a pencil line drawn on a sheetof paper. These have value if the dimensions of a feature are required as adrawing of the image of a stage micrometer can be prepared with the same

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A. J. Lacey

optics. However photomicrographic techniques are widely available andinstruction manuals for the equipment are in the main user-friendly.

9.1 Microscope/camera attachmentsBoth traditional photomicrographic and video camera recording techniquesrequire careful attention to the interface between the camera and the micro-scope to achieve parfocality of the microscope image plane with the plane ofthe emulsion of the film or the chip of the video camera. When the micro-scope is used for visual examination of the specimen the rays of light leavingthe exit pupil of the microscope are almost parallel and the eye is receivingthem in a relaxed state (i.e. focused almost at infinity). The image in visualmicroscopy is said to be a virtual image. Figure 10 illustrates the options that

Figure 10. Images in the eye and for photographic and video camera recording, (a) Imageformation with the eye. The microscope is adjusted so that the primary image falls on thefront focal plane of the eye lens in the eyepiece. This means that parallel rays of light willenter the eye and be focused by it on to the retina. (b) The primary image is lowered byincreasing the specimen-objective distance. This causes rays leaving the eyepiece to con-verge and form a real image in a plane suitable for accommodating the film. But perform-ance of the objective is thereby impaired, (c) Specimen and primary image are in the correctpositions, but the eyepiece has been raised to increase primary image-eye lens distanceand thereby produce a real image on the film. (d) Special projective lens or 'photographiceyepiece' designed to form a real image on the film using the normal settings of the micro-scope. (e) Normal eyepiece and in conjunction with a converging lens in a special photo-micrographic attachment; optically similar to using the eye (a). From ref. 1 with permission.

30


are available for altering the virtual image to make it a real image falling onthe film or chip. The photomicrographic accessories commonly available withmicroscopes (or built-in photomicrographic systems) have auxiliary lenses todo this. You should check the equipment you have available for such acces-sories as projection 'eyepiece', projection lens, or other lenses built-in. Acheck can sometimes be made on the photo equipment by opening the emptycamera and placing a ground glass in the plane of the emulsion when an imagewill be seen. Most microscope manufacturers provide the required adaptorsfor attaching a range of video cameras to their instruments. The video camerais usually fitted with a threaded 'C' lens mount or the bayonet ENG lensmount. Some manufacturers publish a document specifically on the microscopecamera attachments (see for example Olympus 'Video/Photo adapters').Space precludes a lengthy treatment of this subject.

An important piece of the final print is that it contains a scale bar and this isgiven emphasis. Other chapters in the book will contain photomicrographicrecords and the details of the optics used to take them (see for exampleChapter 4, Section 6.3).

Kohler illumination is again important to ensure that the specimen is lit in acontrolled manner, and that the lighting of the background is without distract-ing features. Kohler was himself a photomicrographer and developed themethod of illumination precisely to achieve this control of the light. A singleprotocol is given here for photomicrography and particularly to illustrate theuse of colour filters for black and white pictures (Figure 5) or for the adjust-ment of colour temperature for colour transparencies.

Protocol 10. To make a photomicrograph using (a built-in) 35 mmcamera system

Equipment• Microscope to which is fitted a camera sys- • 35 mm film of known characteristics (i.e.

tern with appropriate microscope/camera B&W or colour, transparency, daylight/interfacing tungsten light, speed)

• Photomicrograph recording sheet Colour contrasty specimen• Range of neutral density and colour filters

Method1. Select the film according to the requirements envisaged (see

Chapter 4).2. Check the camera for dust and load the film.3. Advance the film wind on mechanism checking where possible that

the camera rewind knob is rotating to indicate that the film is beingtransported effectively.

4. Set the film speed (previously verified by experiment) on the controlpanel on the exposure measuring device.

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A. J. Lacey

Protocol 10. Continued5. Prepare the record sheet for notes on each exposure.

6. Clean the specimen slide thoroughly.

7. Set up Kohler illumination, selecting objective and approximate fieldof view of your specimen. Check image is free of blemishes caused bydust in the system.

8. Where necessary ensure that the beam splitting prism in the eyepiecehead of the microscope is directing the light to the camera. Observethe format lines in the new image and adjust the field of view of yourspecimen to the 35 mm format.

9. Defocus the specimen's image and with the eyepieces' focusingdevice critically focus the cross lines (usually seen as double).

10. Return the specimen image to critical clarity by the microscope focus-ing system. This procedure has focused the image on the graticuleand on the emulsion of the film.

11. If using colour film adjust the lamp voltage to the predetermined bestsetting (colour temperature). If necessary, insert any correction filters(i.e. Wratten 80A to compensate for tungsten/halogen lighting whenusing daylight film).

12. Decide whether the exposure meter is required to read the wholefield of view and thereby obtain an average value or, as in the case ofdark ground or fluorescence images, there should be a spot metering.Read the exposure meter and check that it falls in the range of the ofthe equipment.

13. If indicated that the exposure is too short insert neutral density filters.If indicating too long increase the illumination intensity or use fasterfilm possible up to ASA 3200.

14. Again check precision of focus.

15. Press the shutter release avoiding touching any part of the bench ormachine while the shutter is open.

16. Check wind on is working when shutter is closed.

17. Note the details of the optics and specimen on the record sheet.

18. If the specimen is a very important one it may well be useful for aseries of modifications and subsequent exposures made at this point(see for appraisal in step 22).

19. (Optional but valuable.) Take the next exposure with the specimenreplaced by a stage micrometer, keeping all optics as before.

20. For black and white films a series of pictures taken of the stained con-trasty specimen can be photographed using complementary or samecolour filters (see for example Figure 5). There may be considerable

32


changes of exposure necessary (made by the camera control systemif automatic) when combinations of filters are changed.

21. At the end of the film, rewind into the cassette. It is probably falseeconomy to wait for long periods for the film to be entirely used up. Itis better to process promptly even if a few exposures are wasted. Re-cords may be lost, film may not be appropriate for the next occasion,someone else may have taken the film out of the camera! Recordbooks are often used in laboratories but even these may be in-complete, for example they may be without the initials of the lastuser.

22. Critically appraise your pictures looking for excessive warmth (toomuch red) or too cool (too much blue) in your colour transparencies.Uneven lighting will show if Kohler illumination has been poorly set-up, dust in the system will show up. Inaccurate focusing and alsoinappropriate combinations of magnification and numerical apertureof the objective used resulting in lack of resolution of detail or in-adequate depth of focus, all may lead to frustration. Inappropriatesection thickness for the depth of field of the objective may alsocontribute to dissatisfaction.

Protocol 11. To obtain records of an image using a videocamera and video printer, video cassette recorder(Figure 11)

Equipment• Microscope with camera port preferably • Video printer with appropriate paper (B&W

with a trinocular head or colour)• Video camera • TV monitor• Interface accessory for camera to micro- • Stage micrometer

scope (so that the image of the specimen . video cassette recorder (VCR)falls on the chip of the camera)

A. Using a video camera and video printer

1. Fit camera to microscope through appropriate interface. Connectcamera video output to video printer input. Connect video printer out-put to monitor input. Where the TV monitor has a video input im-pedance termination switch this should be set to the 75 ohmsposition. Check paper in cartridge of video printer.

2. Set up Kohler illumination and observe specimen through the eye-pieces.

3. Insert beam splitting prism to direct light to camera.

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A. J. Lacey

Protocol 11. Continued4. Observe monitor. Light intensity will almost certainly have to be

reduced and do this by insertion of neutral density filters. Heat filtersmight also be required to avoid burn out of image on monitor.

5. Adjust field of view portrayed by the monitor and critically adjust focus.6. The frame/field switch would normally be set to 'frame' so that all the

lines of the TV picture are recorded. The 'field' setting produces apicture in which only alternate lines are printed, the scanning timebeen reduced from 40 to 20 milliseconds. This helps to avoid distor-tion or blurring of moving objects in the image. Press print button.

7. Note settings on video printer for such as normal, dark, light (B&Wprinters). The printer's instruction book will help in deciding if there isneed for altering the ex-factory settings after viewing the printedimage.

8. Repeat the procedure for the image of a stage micrometer using thesame optical system. From the print thus obtained place a scale barby hand on to the print obtained in step 7.

9. Some printers have sub-menus for insertion on to the print of detailsof the specimen, optics, etc. This information can be added at step 6.

10. Make photocopies of the print showing the scale bar. These, in somecases, keep more satisfactorily than the prints themselves.

B. Using a video cassette recorder1. Connect the camera output to the input of the VCR. Connect the ouput

of the VCR to the input of the video printer retaining the monitorconnections as before.

2. Audio signals can be fed into the VCR either at the time or in a sub-sequent viewing of the recorded tape. Domestic VCRs may or maynot have a microphone input. If there is no microphone input on theVCR then a microphone amplifier is required as an external accessoryfound in video processors for editing.

3. Again images of the stage micrometer should be recorded.4. Sequences of moving objects can be recorded. Take care not to have

large changes of field of view made too rapidly. Again avoid too longrecordings of situations as viewing subsequently becomes exceed-ingly tedious. Audio record changes of objective, field, specimenchanges, as well as date and name of operator.

5. Prints can be made from the stored images on the tape by switchingoff the camera and using the PLAY mode on the VCR. Use the appro-priate stage micrometer images to insert scale bars on the print asdescribed in part A, step 8.

34

Figure 11. Diagrammatic flow of the information from the light source to the final print (and tape record) in a video printer system. The printis of a specimen of human chromosomes G-banded with trypsin Giemsa stain, courtesy of Mr A. Cuthbert and Dr D. Trott, using microscopeOlympus BH2, objective A40PL, NA 0.65, Hitachi CCD KP113 camera, and Mitsubishi Video Copy Processor, paper K75HM. The inset of thewave form is reproduced from ref. 2 with permission.

A. J. Lacey

Protocol 12a. To make a colour saturation test specimena

Equipment• Microscope preferably with trinocular head • Colour monitor

and appropriate camera microscope inter- . Film base from a processed Polaroidface 'Polachrome' 35 mm film

• Colour video camera Adhesive tape• Colour video printer • Mounting medium such as DPX

Method

1. Take the scrap piece of unprocessed film left at the beginning of aused Polachrome film after processing.

2. Roll the adhesive tape down firmly on to the black side of the film.

3. Peel off the adhesive tape, the emulsion will come away on the tape,leaving the film base with the printed lines on the upper surface.

4. Without turning the film over, mount a small piece of this film on aslide under a coverslip. It is essential that the printed lines are on theupper side of the film, immediately below the coverslip. (The lines areon the less glossy side of the film.)

Note: for reflected light the specimen can be be vacuum aluminiumcoated on the non-emulsion, more glossy side, before mounting onthe slide, without a coverslip.

aThe method for the test specimen is kindly supplied by D. J. Thomson.

Protocol 12b. To test colour saturation of video camera/videoprinter, video monitor

Equipmenta

• As for Protocol 11 but with colour camera, colour video printer, and colour monitor

Method

1. Observe the image of the bands with light set at the correct runningvoltage.

2. Note fully saturated red, blue, and green bands through the eyepieces.Any yellow colour at the junction boundaries of the red and greenbands is an artefact produced by an error in the system such as uncor-rected chromatic aberration in the microscope (or camera optics).

3. Send the image to the colour printer and monitor.

4. Use neutral density filters to correct light intensity. White balance is

36


automatic in most cameras and will compensate for variations incolour temperature allowing the intensity of the light to be controlledalso by the voltage setting on the microscope. Some cameras have a'high speed shutter' which can be switched in for moving material.

5. Enter sub-menu details of optics, etc.

6. Print. Repeat with specimen replaced by stage micrometer if scale baris required to be put on to final print.

7. Visually compare the print with the eyepiece image and/or with a conventional 35 mm photomiurographic image record (obtained perhapsby Protocol 10). Rank your satisfaction level.

The exposure times are automatically determined by the camera factory sellings but by care-ful consultation of the instruction manual can be overridden.

9.2 Summary(l should he remembered that the specimen-light interaction which is por-[rayed in the microscope image is changed by a photochemical process in thephotographic system into silver grains or dye particles impressed in paper.

Figure 12. Video print of a 'Polachrome'35 mm film base (see Protocol 11). Olympus BX50 microscope, UPLANFI, •/-,'. 17, \ 40/0.75 objective, JVC colour video camera TK-128OE,Sony Mavigraph UP 1200 EPM video printer. Width of green band 7/jun.

37

A. J. Lacey

The changes in the chemistry are then chemically fixed. This pattern ofchange is then viewed by the observer's eyes in light reflecting prints or lighttransmitted in transparencies. In the case of the video process the image ischanged into a pattern of electronic charges which are read directly, in ananalogue system, or translated into a digitized format in the digital camera ordigitizer. The signals are then changed back into light and darkness patternsby a further process of electrons interacting with phosphors in the TV tube.The response in these phosphors is what the eyes see and interpret. Or altern-atively the electrons cause a change in reactive paper or cause sublimation ofdyes onto paper—the video print. The resulting patterns on the print areagain interpreted as patterns of light and dark (or colour) seen by the eye.

The relative ease of obtaining a print from the video printer techniquewhether direct from the camera or from a previously recorded image is veryattractive. The economics of the video system compared to that of dark-roomaided photography must be a factor in deciding which type of situation alaboratory might choose. The archiving of prints is probably much the same inboth cases. The use of cine film for recording moving images is now virtuallyobsolete. The transfer, however, of achival cine records to video tape, bestdone commercially, is very satisfactory.

Protocol 13. Preparation of onion skin peels for comparison ofcontrast techniques (5 with permission)

Equipment• Inner bulb scales from fresh onions • 1 cm cork borer• 0.5 mm plastic sheeting (easily obtained • Scalpel and artist's water colour paint

from empty storage bottles for non-toxic brushchemicals) . A range of microscope contrast techniques

• Double-sided adhesive tape (Agar Scientific) available preferably on the same machine

A. Preparation of onion skin peels

1. Cut the plastic sheeting into 15 mm X 15 mm pieces. Remove a centralinner core with the cork borer, leaving a plastic holder with a hollowcentre.

2. Firmly secure one side of the holder to double-sided adhesive tape,trim the tape flush with the edges of the holder, and remove the tapecovering the central hollow by cutting round the edge of the hole witha sharp scalpel.

3. Obtain the inner bulb scale of the onion avoiding wetting the surfaceof the inner epidermis with sap.

4. After removing the remaining protective layer of the adhesive tape,press the plastic holder firmly against the inner epidermis of the scale.

38


Cut the epidermis with the scalpel around the outer margin of theholder.

5. Lift the holder off which will remove the epidermis adhering to theplastic holder. Handling the holders by the edge with forceps willprotect the epidermis from damage.

6. Store the specimens in water in a Petri dish until required. Any gelatin-ous cell wall material adhering to the side of the epidermis in contactwith the water may be wiped off with the soft paint brush.

7. Mount a holder in water on to a slide. Cover with a glass. Go tocontrast techniques.

B. For short-term preparations

1. Isolate by a series of shallow cuts a piece of the inner epidermis of aninner scale of an onion bulb. Grip one edge with forceps and strip fromthe scale.

2. Mount in water on a slide, taking care to keep the outer surface of theskin upwards in order to maintain its orientation to the light and to theeye. Cover with a coverglass. This preparation will last for a consider-able time particularly if kept moist and cool.

C. Contrast techniques

1. Examine the specimens under each type of contrast technique includ-ing transmitted and incident light for bright-field, dark-field, phase-contrast, polarized light (single polarizer and crossed polars), DIC, andepifluorescence (auto and with fluorochromes). Figure 9 shows theappearance of the epidermis under five of these contrast techniques.

2. Prepare a chart such as in Table 2 giving sufficient space for commentson the image of the various techniques utilized.

3. Enter into the chart the levels of brightness or relative intensity of thevarious features and their backgound. Remember to enter equalbrightness or darkness if there is no contrast between features.

4. You may wish to amend the entries given in the Table 2 but beprepared to think through why you disagree with what is entered.

Notes: for quantitative data on such features as the mass of the nucleus orthe refractive index of the cytoplasm then immersion refractometry usingphase-contrast or Jamin-Lebedeff interference microscopy could be tried.Relative dimensions and volume can be obtained by reference to techniquesdescribed in Chapter 8. Plasmolysis of the cytoplasm can be achieved byimmersing the epidermis in 2% sea salt in water which has a higher osmoticpressure than the vacuolar sap.

39

Table 2. Onion skin examined with various microscope techniques

Technique Background Onion skin components (see Figure 13)

Nucleus Cytoplasm Vacuolea,b

Light-matter interaction

Phase(+ve) Brightgreen filter green

DIC Bright

Walls

(%.13/4 (Fig. 13/5and 4a) and 61

Horizontal

Top Bottom

Transmitted

Bright-field Bright Only visible with aperture closed down (rotten image throughout)

Dark-field Dark Sky grey Granules sky grey or bright

Polarized light Bright(plane)

Polarized light Dark(plane)with crossedpolars

Crossed polars Red one colour+ sensitive tint

Dark with halo Dark granules with haloesround edge Movement

Visible Granules clearly visible(3D effect) Movement

As bright-field

Very slight white

Vertical

Short Long

Bright in all orientations

Bright Bright(on either side of dark line)

Diffraction effects and absorption map

Scattering by reflection and refractionBoundaries of refractive index

Map of optical path in z direction = t(no - nm)

Bright Bright Map of optical path as above (but(but see under polarized light) with boundaries accentuated by 3D effect)

No change on rotationof specimen

No pleochroism apparent

Bright Bright Map of birefringence, o.p.d.(at certain orientations only) and orientation = t(n11 - n1)

Blue Yellow(or yellow) (or blue)

Indicate direction ofbirefringence ±(n11-n1)

AVEC-DIC

Fluorescence see under incident fluorescence

Confocalc

Incident

Bright-field Bright

with surface replica and sputter coatingDark-field Dark

FluorescencePrimary Dark

Transvacuolar strandsand ERd

FluorescenceAcridineOrange Dark

Fluoroprobes Dark

Confocalc

Blue Yellow See DIC above but with enhanced contrast(or yellow) (or blue) Indicates direction of birefringence ± (n11-n1)

Some brightness but excessive Visible(?) Map of reflectivenessglareNegative or positive of surface characteristics depending on single or double replicationClear contour of brightness Grades into horizontal walls Map of surface light scattering features

Dark channels in wall clear

No primary fluorescence apparent

Apple green

Various colours depending on probes and irradiating wavelengths(Vacuole visible with LYCHb)

(If over-stained yellow green) Map of chemical affinity for Acridine Orange

aThe vacuole can also be inferred by achieving plasmolysis when mounting medium such as 2% sea salt in water has a higher osmotic pressure than the cell sap.b Oparka et al. (4) has described the use of LYCH on onion preps.c Confocal microscopy is clearly potentially very powerful for optically sectioning living onion cells.d Video microscopy using VEC and VIM (3).

A. J. Lacey

42


Figure 13. Diagrammatic three-dimensional appearance of onion epidermal cell in itsrelation to the light in a microscope and the observer's eye. (a) How the cells appear seenfrom the top (x and y directions) (see Figure 9). 1. Vertical walls in the (z) axial plane (a)short and (b) long. 2. Horizontal wall in the plane of the stage of the microscope. Upperwall, (a) upper surface (b) inner surface. 3. Lower horizontal wall, (a) inside cell surface (b)outer. 4. Nucleus with (4a) nucleolus. 5. Cytoplasm with granules (with insert courtesy ofDr N. Allen and ref. 3 with permission sought). 6. Vacuole.

Figure 13 gives a three-dimensional impression of what the epidermal cellscan be interpreted as being like after examination with a wide range of tech-niques. The scientific literature is adding information continuously to thisimpression of the physicochemistry of onion epidermal cells. Video micro-scopy from the laboratory of Professor Allen (3) and fluorochrome studies byDrs Oparka and others (4) have greatly expanded the information on thevacuole and its contents.

AcknowledgementsInevitably in this introductory chapter or summary of basic microscopy thereare a number of points which were derived from colleagues and teachers overthe years and their contribution is hereby gratefully recognized. Especialthanks for his reading the whole script and amending the text and the proto-cols on the video recording go to Mr D. J. Thomson. Thanks too to Dr PeterEvennett for permitting his figures to illustrate much of the chapter. Thanksare due for the protocol on preparation of the onion epidermis given by DrOparka and for the illustration of the details of the endoplasm to Dr NinaAllen. Colleagues Drs Cuthbert, Trott, and Jobling have kindly allowed theauthor access to their video printers.

References1. Evennett, P. J. (1989). In Light microscopy in biology: a practical approach (ed. A.

J. Lacey), p. 61. IRL Press, Oxford.2. Trundle, E. (1987). Newnes television and video engineer's pocket book. Butter-

worth Heinemann, Oxford.3. Allen, N. S. and Brown, D. T. (1988). Cell Motil. Cytoskel, 10,153.4. Oparka, K. J., Murant, E. A., Wright, K. M., Prior, D. and Harris, N. (1991). J. Cell

Sci., 99, 557.5. Oparka, K. J. and Read, N. D. (1994). In Plant cell biology: a practical approach

(ed. N. Harris and K. J. Oparka), p. 27. IRL /OUP Press, Oxford.

43

Introduction to confocal microscopyP. J. SHAW

1. IntroductionOptical microscopy is inherently limited in resolution by the wavelength of thelight used in the technique; in practice this resolution limit is generally about0.25 jjim. However, there are several factors which make optical methods verypowerful. One major advantage comes from the combination of microscopywith fluorescent probes. Almost any biological component can be specificallylabelled with a fluorescent tag, sometimes called a fluorochrome, and thenimaged by virtue of its fluorescent light emission. The available fluorescentprobes include dyes for specific components, such as nucleic acids, reportersfor particular ions such as calcium, modified DNA and RNA sequences, whichare used for fluorescent in situ hybridization, and antibodies raised againstalmost any biological component imaginable. Fluorescent detection has theadvantage of great specificity and sensitivity. Optical filters are used to providelight of just the wavelength needed to excite the fluorochrome, and a dichroicmirror and an emission filter can then be used to provide an image arising justfrom the fluorescently emitted light. This has the effect of a high degree of dis-crimination against the illuminating wavelength and in favour of the fluores-cent tag, resulting in a very good signal-to-background ratio. This selectivity iseven greater in the nearly universal epi-illumination configuration; the imagingobjective lens is also the illuminating condenser lens, and so in this arrange-ment the illuminating light which has not undergone absorption passes throughthe specimen and cannot contribute to the image background. A second majoradvantage is that light is relatively non-destructive to biological material, andso with care and skill optical microscopy can be used to image living cells, tis-sues, and organisms without too much damage. Many studies over the past fewyears have used fluorescence microscopy in living cells. In some cases fluor-escent reporters can be simply perfused into living cells—for example, AM(acetoxymethyl) esters of calcium-sensitive dyes. Cellular esterases cleave theester linkage, and prevent the dyes from diffusing out again. In many othercases microinjection has had to be used to introduce fluorescent probes. Forexample, a purified protein can be covalently labelled with a fluorochrome andthen microinjected into cells. In favourable cases, the introduced protein will

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behave in the same way as the native protein, allowing fluorescence micro-scopy to be used to determine its location and dynamic behaviour within theliving cell. Most recently molecular genetic techniques have begun to be usedto link green fluorescent protein (GFP)—a naturally fluorescent protein foundin a jellyfish—to proteins of interest. A cDNA is constructed containing thetarget protein and the GFP sequence, under the control of a suitable pro-moter, and is then transformed either transiently or stably into the cells ororganisms of interest. In a surprising number of cases the extra protein domainof the GFP does not seem to interfere with function and location of the targetprotein, and thus cells can be made which directly express a fluorescent ana-logue of the protein of interest. This approach is already beginning to bringabout a revolution in the cell biological and developmental study of livingorganisms and cells.

2. The problem of out-of-focus lightIn spite of its advantages, conventional optical microscopy has some trouble-some features. In principle if all the fluorescent light emitted from a specimencould be recorded, it should be possible to reconstruct a perfect map of thedistribution of the fluorochromes which emitted the light, at least to the reso-lution limit specified by the wavelength. In practice this is not the case. Thereason lies in the geometry of a practical microscope. The emitted light mustbe collected by passing through an objective lens, and this of necessity intro-duces an aperture into the system. The result is that some of the emitted lightcannot be recorded, and the image reconstructed by the optical system has theresolution seriously degraded, particularly in the direction of the optical axis(usually denoted z). Furthermore the degradation depends on the level ofimage detail in a rather complicated way. Thus fine details, often called highspatial frequencies, are relatively little affected, whereas large structures, orlow spatial frequencies, are greatly spread out in the z direction. This is oftencalled the problem of 'out-of-focus' light. A small sphere ends up beingimaged in three dimensions as a complicated elongated structure. This is dis-cussed in more detail below. Some interesting approaches to this problemattempt to measure more of the sphere of light emission and thus to produce areconstruction which suffers less from these defects (1), but for the purposesof practical biologists there are two feasible current methods to overcome theproblem. The first one is confocal microscopy; the optical system is modifiedso as to minimize the problem by eliminating or at least reducing the contribu-tion of the out-of-focus light. The second approach is to measure the conven-tional image accurately, out-of-focus light included, to measure also theproperties of the imaging system in detail, and to use image processing tech-niques to remove the out-of-focus contribution from the image. This chapterwill attempt to describe in as non-technical a way as possible the basis ofconfocal microscopy. There has been much published in recent years about

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2: Introduction to confocal microscopy

confocal microscopy. One of the best sources of information is the secondedition of the Handbook of biological confocal microscopy (2).

2.1 The confocal principle: explanation by ray opticsThe most common way to explain the operation of a confocal microscope isby a ray diagram similar to the one shown in Figure 1. A light source, almostalways a laser, is used to provide an effective point source illuminationthrough a pin-hole. At the specimen this gives the diffraction limited image ofa point. With a conventional light source this would be the Airy pattern. Witha laser beam the focus is somewhat different, but may still be thought of asapproximating an Airy pattern. Some of the emitted light which originates fromthis plane passes back up through the objective lens, through the dichroic mirrorand emission filter, and through a second pin-hole, finally being detected by aphotomultiplier behind the detector pin-hole. The two pin-holes—illuminatingand detecting—are both located in planes conjugate to the plane of focus in

Figure 1. Ray diagram of confocal optical arrangement, showing how light rays originat-ing away from the plane of focus are eliminated from the image by the detector aperture.

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the image ('confocal' is derived from a contraction of 'conjugate' and 'focal').However, the out-of-focus problem arises because parts of the specimenabove and below the plane of focus are also illuminated and also emit light.Now consider rays of light originating from above the plane of focus (the lightshaded rays in Figure 1). Originating nearer to the objective lens, these raysare brought to a focus further away from the objective on the other side,behind the detector pin-hole. At the level of the detector pin-hole, these rayshave not converged sufficiently to pass through the pin-hole, and so they areeliminated from the resulting image. A similar argument applies to light fromparts of the specimen below the plane of focus. Therefore the confocalarrangement—the interaction of the illuminating and detecting pin-holes—has the effect of discriminating against light originating away from the planeof focus.

So far, we have described confocal imaging of a single point on the speci-men. For an imaging system, a method is needed to image successive points tobuild up an image. This is done in several different ways in different designs,and is one of the main differences between the different types of instrument.This is discussed in more detail later.

2.2 Linear, shift-invariant imaging and the point spreadfunction

The explanation given above uses a ray approximation, and while it explainsin general terms the exclusion of out-of-focus light, it does not provide a verydetailed idea of the image that a confocal microscope will produce. Wegive next a somewhat better approximation, although still simplified; a fullexplanation would inevitably be very mathematical.

First, we will consider a microscope, confocal or conventional, in systemengineering terms as a device which takes an input—the specimen—andtransforms it to an output—the image. Now imagine cutting the specimen intotwo pieces, and imaging each piece separately. If adding these two imagestogether produces the same result as imaging the whole specimen, the processis described as linear; the whole is the sum of the parts. If this condition holds,as it does for many types of imaging, including fluorescence microscopy, thenthe specimen can be imagined to be cut into smaller and smaller pieces. Ulti-mately the pieces can be made arbitrarily small and thus can be considered aspoints. Then the image of the specimen is simply the sum of the images of allthe individual points. In principle the image of a single point therefore pro-vides all the information needed to characterize the imaging process. Thisimage of a point is usually called the point spread function (p.s.f.), and mathe-matically the operation of replacing each point by the point spread functionand adding the resulting point images together is called a convolution. In sum-mary, we can describe a linear image formation process as the convolution ofthe specimen with a point spread function. A further question is whether the

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point spread function is the same for every point in the specimen. If it is thesame everywhere the imaging is said to be shift-invariant. Whether this con-dition holds is not always an easy question to answer. It is certainly a goodapproximation for fluorescence imaging, but is discussed in more detail below.In fact this explanation is not limited to optical microscopy. It applies to manyother types of microscopy, to astronomical imaging through telescopes, tovarious sorts of spectroscopy, and many other biomedical and physical imagingmethods.

Thus in conventional fluorescence microscopy each point of the specimen isreplaced by a copy of the p.s.f., whose intensity is directly proportional to theintensity of the point. In a confocal microscope, the same argument showsthat the illuminating pin-hole, effectively a point source, produces a light dis-tribution at the specimen which is also given by the p.s.f. Light is detectedfrom the specimen only if it passes through the detector pin-hole. In principlethis is equivalent to a point detector, although in practice most confocalmicroscopes allow the detector pin-hole to be opened to admit more light,which causes a departure from true confocal imaging. Since light paths arereversible, this means light is detected from a region of the specimen corre-sponding to the image of the detector pin-hole at the specimen—again givenby the p.s.f. Thus the overall effect is that the p.s.f. is applied twice in confocalimaging—once because of the illuminating pin-hole, and again because of thedetector pin-hole. As far as the detected confocal image is concerned, applyingthe p.s.f. twice means that the effective confocal p.s.f. is the original, conven-tional p.s.f. squared. Since the p.s.f. comprises a central maximum with lowersurrounding maxima, squaring it reinforces the central peak while weightingdown the surrounding components. It is the subsidiary maxima that give riseto the out-of-focus contribution in conventional microscopy, and so decreasingtheir relative weight in the confocal p.s.f. has the effect of removing a largepart of the out-of-focus light.

2.3 The shape of the point spread functionSo far, we have left an explanation of the actual shape of the p.s.f. somewhatvague. There are two ways to determine the form of the p.s.f.: we can derive itfrom optical theory or we can actually measure it by using a specimen which isas near as possible to a point. A full derivation of the p.s.f. requires physicaland mathematical theory which is beyond the scope of this chapter, and in anycase probably no theoretician has yet adequately taken account of all the var-ious aberrations and other factors which are necessary to derive an accuratep.s.f. for a real microscope. On the other hand, an outline explanation may behelpful, so we shall give one; some measured p.s.f.s are shown below.

Light can be described as waves. A simple, but very useful way of understand-ing the light originating from an object was first given by Huygens. Accordingto this principle, each point on an object acts as a secondary point source for

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light waves. In the case of fluorescence, each fluorochrome molecule will besuch a point source. Thus the total light emission consists of many sphericalwaves expanding from the points. Within certain limitations, which need notconcern us here, this sum of spherical waves can be described as the Fouriertransform of the object. In fact, what is usually called the diffraction pattern isactually the intensity of the Fourier transform, and this is a simple way tounderstand Fourier transforms—a pattern derived from the image where lowresolution components, often called low spatial frequencies, are near the originat the centre of the pattern, and high resolution components, or high spatialfrequencies, are furthest away from the centre towards the edges of the pattern.In order to change back the emitted waves to an image, an inverse Fouriertransform is needed—this is exactly what an optical lens does. However, notall the light coming from the object enters the lens—there is inevitably anaperture within which light is passed, and outside of which light is excludedfrom image formation. This aperture, wherever it is actually located in a lenssystem, may be regarded as an aperture in the diffraction or Fourier transformplane. It is generally called the back focal aperture of the lens. The effectiveradius of this aperture depends, among other things, on the numerical apertureof the objective lens. Thus the subsequent image made by the lens—theFourier transform of the diffraction plane—is modified by the aperture.Rather than show the effect of this mathematically, we refer interested readerselsewhere (3), and simply demonstrate this result by a computer simulation.Figure 2A shows a point, corresponding to the original object. The Fouriertransform of a point is a flat, uniformly grey plane (Figure 25). We next passonly components within a circular aperture (Figure 2C), and then carry out asecond Fourier transform of these components. This gives the pattern shownin Figure 2D. This is a circular central maximum, surrounded by annularregions of light and dark. This is in effect the Airy pattern, and it is producedby the circular aperture mask within the lens system.

The three-dimensional p.s.f. is more complicated. The central disc of the Airypattern becomes weaker away from the plane of focus, and the concentric ringsbecome relatively stronger and increase in diameter. This is shown by imagescollected from small fluorescent beads in Figure 3A. (See Chapter 6, Protocol2for a suitable specimen preparation method.) These out-of-focus bead imagescan be thought of as focal sections through a 3D p.s.f., in which the ringsactually comprise a series of concentric cones. This is shown in Figure 3C inwhich a 3D p.s.f. has been resectioned so as to show a central section parallel tothe optical axis (an x-z section). The diverging cones are seen as diagonal brightlines radiating out from the central maximum. The equivalent measured p.s.f.from a confocal microscope is shown in Figure 3B. The main difference isclearly that the subsidiary maxima have been much reduced in comparison tothe central maximum as theory predicts. When resectioned as a central x-zsection (Figure 3D) the overall pattern is much closer to an elongated ellipsoid,with little contribution from the subsidiary rings.

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Figure 2. Computer simulation of the effect of a circular aperture in the diffraction planeon the image of a point object. IA) A single point source is shown. (B) Its Fourier trans-form is a uniformly grey plane. (C) The finite numerical aperture of the objective lensgives rise to a circular aperture in the Fourier or diffraction plane. (D) The inverse Fouriertransform carried out by the optical action of the objective lens then produces the Airypattern of concentric rings surrounding a central maximum.

2.4 Aberrations and the limits to linear, shift-invariantimaging

In the absence of aberrations, the p.s.f. should he circularly symmetric aboutthe z axis, and should have reflection symmetry about the x-y plane, i.e. itshould be the same above and below the plane of focus. It is clear that themeasured p.s.f. shown in Figure 3A is different above and below the focalplane, and this is due to the presence of aberrations, mostly spherical aberra-tion. It is very common in fluorescence microscopy to observe the presence of

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Figure 3. Measured bead images of subresolution fluorescent beads to show the form ofthe 3D p.s.f, (A) Series of optical sections through a subresolution fluorescent bead,imaged using a cooled CCD camera and conventional fluorescence microscopy. Noticethe concentric rings expanding away from the centre away from the in-focus image(which occurs on section nine in the series). The optical sections are spaced 0.4 urn apart.(B) Equivalent series of optical sections through a subresolution fluorescent beadcollected by confocal microscopy. The out-of-focus rings are virtually absent. (C) Theconventional fluorescence data shown in (A) has been enlarged and resampled in the x-z

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spherical aberration. To see this in any fluorescence microscope, simply find asmall, bright structure on a fluorescence specimen and observe the way itsimage changes above and below the plane of focus. Almost invariably, theimage will disappear much more quickly on one side of focus that the other.This is true even with high quality, well corrected objectives, and arises becausethe objectives are usually not used for real specimens under the conditions forwhich they were designed. For example, oil immersion objectives are usuallydesigned to image objects immediately underneath a coverglass of the correctthickness (usually 0.17 mm), through the immersion oil. Generally, however,the interesting part of the specimen is further away, and so light also passesthrough the intervening mounting medium (usually water or a glycerol:watermixture) which has a different refractive index from the immersion oil. Thisrefractive index mismatch introduces spherical aberration. It also causes an'apparent depth' effect, so that the physical changes in the objective or stageposition used for focusing are not the same as the changes in the plane offocus—usually the change in focal plane is 10-20% less than the physicalfocus movement. These effects are minimized if the immersion medium forthe objective is the same as the mounting medium, and several companies arenow selling water immersion objectives for imaging cells in water-based media.However, this is not a complete answer because cells and cellular constituentsthemselves have a range of different refractive indices—for example, nuclei,starch grains, and plant cell walls have high refractive indices. The effect ofthis is that the focal plane is not really flat, but has bumps in it correspondingto the different refractive indices in the structures. The spherical aberrationcaused by refractive index mismatch is likely to be dependent on the depth ofthe focal plane within the specimen. Therefore, the imaging is no longer shift-invariant, since the p.s.f. differs for different z planes within a 3D image.

Chromatic aberration can also have important effects, particularly in con-focal imaging. In the presence of chromatic aberration, light of differentwavelengths is brought to a focus at different focal planes, and in the case oflight originating away from the central optical axis, at slightly different x-ypositions. We have shown above that confocal image formation can be con-sidered as the overlapping of the images of the excitation and detection pin-holes. For fluorescence imaging, the excitation light is a shorter wavelengththan the detected light, and if chromatic aberration is present, the two pin-hole images are centred at slightly different places and so do not overlap fully.

plane to show the 3D shape of the p.s.f. more clearly. The out-of-focus rings (or cones in3D) can be seen to be much stronger one side of focus than the other, due to sphericalaberration. (D) A similar x-z section of the confocal p.s.f. shown in (B). These images wereall measured using a Leitz planapo x 63 oil immersion objective (NA 1.4). The micro-scope was a Zeiss photomicroscope, either linked to a Bio-Rad MRC600 confocal micro-scope, or to a Photometries series 200 cooled slow-scan CCD camera. The beads werelabelled with fluorescein, and imaged with standard fluorescein filter sets in each case.Laser excitation wavelength 488 nm.

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This reduces the amount of light detected and can change the effective shapeof the confocal p.s.f. Moreover these effects are position-dependent, the over-lap becoming worse the deeper into the specimen or the further the imaging isfrom the optical centre. Thus, again, the p.s.f. is no longer shift-invariant.

The significance of different types of aberration on confocal imaging is stilla matter for research and debate. It is clear that the detected confocal image,even with visible wavelengths, becomes increasingly faint deep into thickspecimens (say 100-200 mm). This effect is most marked for shorter wave-lengths, and least for longer wavelengths. It is probably due to a combinationof increasing aberrations and increasing light scattering.

Chromatic aberration is of very great importance for UV confocal imaging,since virtually all achromatic or apochromatic objectives are corrected onlyfor some visible wavelengths, and have considerable achromaticity in the UV(in fact very few objectives even transmit very well below 400 nm). This hasmeant that confocal microscope manufacturers have had to make specialallowances for UV imaging. In the machine made by Leica (TCS UV), theilluminating pin-hole for the UV channel is placed in a different plane fromthat for the visible excitation channels. In the case of the Bio-Rad UV design(MRC 1024 UV), which has a single illuminating aperture for all wavelengths,a pre-focusing lens system is used in the UV light path, so as to bring the focalplane for the UV light to the same level as the other wavelengths. A similarcompensation is used in the machine made by Zeiss (LSM 510-UV). (SeeHandbook of biological confocal microscopy (2), Chapter 27 for a detaileddiscussion of UV confocal microscopes.)

3. Practical implementation of confocal scanningsystems

We have shown how the confocal pin-holes combine to eliminate the out-of-focus light from detection for a point on the specimen and thus improve thecharacteristics of the p.s.f. To complete the description of a practical confocalmicroscope, we now consider in outline how the image of a specimen planeis built up by a scanning mechanism. The third dimension (z) for a three-dimensional image is produced by scanning 2D images at successive focalplanes, either by moving the specimen stage or the objective through a seriesof small focus steps.

3.1 Point scanningIn principle, the illuminated spot can be itself scanned across the specimen ina raster, in a manner very similar to the scanning electron beam in a TVscreen, or the specimen can be moved through a stationary light path. The latterdesign has the advantage of a simple and accurate optical design, but suffersfrom lack of speed in scanning an image, particularly at low magnification.

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Some of the first confocal microscopes built were of this stage scanning type.However, virtually all current biological confocal microscopes use a scannedbeam design. An angular deflection of the light beam at a diffraction planebecomes transformed into a translation in the specimen/image planes. Thus, asystem of two deflecting mirrors scanning back and forth about two axes posi-tioned at or near a diffraction plane can be used to scan the light beam in a 2Draster across the specimen. The scan driving and measurement circuitry areinterfaced together so that light intensity measurements are taken whichcover the specimen area in a regular raster, and these intensities are digitizedinto a computer image framestore to produce a digital image. Generally imageaccumulation and averaging are provided, either frame by frame, or line byline, or both. Often, the scanning rate can also be changed.

This principle is used in confocal microscopes made by several companies,including Bio-Rad, Leica, and Zeiss. With currently available mirror deflec-tion systems, the maximum scanning rate is a few frames/second (i.e. wellbelow 'real time' or video scanning rates). A detailed description of the vari-ous scanning systems and a discussion of their relative merits is given byStelzer (Handbook of biological confocal microscopy (2), Chapter 9). We willnot attempt to give a detailed discussion of the merits of these machines, sincethe models are still evolving and so such data would quickly be out of date.Most of the spot scanning confocal microscopes currently in use are fromthese three manufacturers, and anyone considering purchasing a confocalmicroscope is advised to test at least these machines. It is important to use aspecimen which is stable and familiar for assessment, so that the user knowswhat sort of image to expect, and preferably can image the same specimen onall the contending machines. It is also a good idea to take images in stored dig-ital form away from all the tested machines to compare with each other on aknown computer and printer back at the home laboratory.

3.2 Slit scanningIn order to increase the scanning to video frame rates or higher, slit scanningdesigns have been developed. Instead of a pin-hole aperture giving a diffrac-tion limited spot, a narrow slit of light is scanned in a direction at right anglesto its length across the specimen. This is achieved in a similar way to spotscanning by using one or more scanning mirrors and a stationary slit aperture.The emitted light is then passed through a narrow detector slit. This has theadvantage that since only a one-dimensional scan is required, the scanningrate can be much faster than in a point scanning system. Furthermore, since aline of the specimen is imaged at one time, the rate of light accumulation fromthe specimen is much higher. However, the disadvantage is that a proportionof the out-of-focus light—that component which is distributed in the directionof the slit—is also detected, and so the optics are only partially confocal. Thisdesign, therefore, is inherently not capable of producing such clean opticalsections as a point scanning system, but the fast scanning speed and bright

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image produced mean that the image can be observed directly through aneyepiece, as in a conventional microscope. For many applications, particularlythose where a single focal section relatively free from out-of-focus blur isrequired rather than a complete 3D image, this design provides a good com-promise between conventional imaging and confocal imaging. Linked up witha high sensitivity CCD (charge coupled device) camera, it could be a goodchoice for dynamic studies of weak or light-sensitive specimens. Slit scanningmicroscopes for biology are currently made by Bio-Rad (ViewScan) andMeridian Instruments (Insight). Slit scanning and video rate systems arediscussed in detail in the Handbook of biological confocal microscopy (2),Chapters 25 and 29.

A design which is intermediate between a point and slit scanner is made byNoran. This machine, Odyssey, scans the incident light beam with a devicecalled an acousto-optical modulator (AOM). AOMs work by using soundwaves to set up a standing wave in a crystal, which then behaves in a verysimilar way to a diffraction grating, producing a diffracted beam at an angle tothe incident beam. By modulating the sound frequency and thus the effectivegrating spacing, the beam can be deflected or scanned through differentangles. The major problem is that since the deflection is wavelength-dependent,unlike a mirror system, the fluorescent light, at a longer wavelength than theexcitation light, cannot be sent back along the same path through the AOM.In practice the detected light is passed through a slit aperture. Thus the opticalarrangement is point scanning for excitation, slit scanning for detection. Thedesign is capable of very high scanning rates.

3.3 Spinning discThe final confocal design we shall describe was actually the first to be built, byPetran (4). Instead of a single pin-hole and a point light source, this designuses an extended light source and an array of many pin-holes on a Nipkowdisc which is placed in a conjugate image plane. In the original design thearray of pin-holes was symmetric about the centre of the disc. The incident lightpassed through each pin-hole, and the reflected or fluorescent light passedthrough equivalent pin-holes on the opposite side of the disc. In a more recentdesign, the emitted light passes back through the same pin-holes. The con-dition necessary for confocal imaging is that no emitted light should passthrough the 'wrong' pin-hole, and this means that the pin-holes must bespaced far apart relative to their diameters. This means that only a limited setof points are imaged by the disc in each position. The full image is obtained byspinning the disc rapidly, so that the pin-holes, which are usually arranged in aspiral pattern, scan across the whole image area. The main problem with thisdesign is that the light source has to be spread out over the whole of the disc,and so is orders of magnitude less bright that the single pin-hole/laser arrange-ment. Furthermore, only a very small proportion of the available light passes

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through the disc to illuminate the specimen, and more seriously, only a verysmall proportion of the reflected or emitted light passes back through the discto be detected. The result is that it is difficult to record enough light for asatisfactory image, particularly in the case of fluorescence, and much of theavailable fluorescent light is wasted. This has substantially limited the use ofthis design in biological applications.

Juskaitis et al. (5) have recently designed a variation of the spinning discapproach which overcomes the problem of poor efficiency to a large extent. Adetailed description is beyond the scope of this chapter, but in essence theyhave shown that it is possible to design discs with 25% or more light transferefficiency that produce an image which is the sum of the conventional andthe confocal image. In the prototype machine, half the disc was of this design,the other half was transparent. The image produced therefore alternatedbetween the conventional and the composite image. Digital image processingwas used to subtract the conventional from the composite image, leaving theconfocal image. It is hoped that this simple and elegant idea can be quicklycommercialized; it promises to be a very cheap, powerful, and efficient system.

3.4 Two-photon imagingTwo-photon imaging has attracted much attention recently. So far, severalresearch machines have been constructed, and a commercial system is avail-able from Bio-Rad. In conventional fluorescence microscopy, the specimen isilluminated with photons of the correct wavelength to raise an electron in thefluorophore to a higher energy level. However, if a high enough intensity oflight at double the required wavelength can be used, then the fluorophore canabsorb two photons almost simultaneously to produce the required energylevel change. The combined probability of the double absorption depends onthe square of the light intensity distribution. Thus, the illuminating p.s.f. is thesquare of the conventional p.s.f. (i.e. very similar to a confocal p.s.f.). In prac-tical instruments, a detector pin-hole has also been used to give an improvedp.s.f. The advantages of this method are that light should only be absorbednear the focal spot, and thus should only cause fading and photodamage atthis position in the specimen. In normal confocal imaging, although light isonly detected from near the focal plane, light is absorbed by the specimen atevery plane, causing the associated photobleaching and other photodamage,in just the same way as in conventional, wide-field microscopy.

Another potential advantage of two-photon imaging is that the long illumi-nating wavelengths used—about 1000 nm—are relatively good at penetratinginto deep specimens. Also, UV fluorophores can be excited with visible wave-lengths, which avoids the problems mentioned above with UV optics. The dis-advantage is that a powerful and expensive pulsed laser system must be used.Currently, such laser systems cost in the region of £100000, but it is hopedthat more affordable lasers will soon be available. Another potential problem

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is damage to the specimen by the high intensity long wavelength incident light.Experience with different cells is so far limited; some living cells have beenimaged without problem, but the presence of any cellular constituents whichabsorb in the far red/near IR region of the spectrum would have disastrouseffects, and cause the specimen to be rapidly destroyed by heating.

4. Comparison of conventional, wide-fieldfluorescence imaging with confocal fluorescenceimaging

We refer readers elsewhere to a more detailed comparison of wide-field andconfocal imaging (6), merely giving here a brief discussion of some of themore important aspects of this topic. A major limitation of most current con-focal microscopes, apart from the spinning disc designs, is that they must uselaser light sources, with consequent severe restrictions on the available wave-lengths. While developments in laser technology will reduce these limitations,it is unlikely that they will disappear in the foreseeable future. In contrast,conventional, wide-field fluorescence microscopy can use virtually any wave-length from the visible spectrum and beyond.

4.1 Noise and resolutionThe major advantage of confocal microscopy over conventional, wide-fieldmicroscopy is the elimination of the out-of-focus light. This is broadly equiva-lent to an increase in resolution in the z direction—the optical axis. In prin-ciple, a confocal microscope is capable of better resolution in the image (x-y)plane as well, but this will only be realized if very small pin-holes are used,and if the signal-to-noise ratio is large enough. Figure 4 shows a diagram ofthe contrast transferred (in the x-y plane) as a function of resolution for awide-field and for a confocal microscope (data redrawn approximately fromref. 7). It is clear that the confocal microscope has a better contrast transfervalue, especially towards the resolution limit, than the wide-field microscope.However, the image detail actually observable will also depend on the noiselevel in the image; the higher the relative noise, the more the image componentswill be lost beneath it. Since the image contrast decreases at high resolutionthis means that increasing the noise level will have the effect of decreasing theresolution in the image; coarse, large scale structure remains above the noise,while fine detail is lost. In practice, the noise in a confocal image can easily bevery much higher than the noise in a wide-field image as we will show below,and so it is entirely possible for the effective resolution to be worse for aconfocal image.

For the sake of this discussion we will assume that a wide-field image isrecorded with a high quality, scientific grade cooled CCD camera, and com-pare it with the image from a spot scanning confocal microscope which uses a

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photomultiplier detector (see refs 6, 8, and 9 for more detailed discussions).Leaving aside for the moment where in the specimen the photons making theimage have come from, we will compare the two systems purely in terms oftheir image detection efficiency and noise. Noise in the image can beconsidered as coming from two sources:

(a) Detector noise—noise introduced by the detection device and circuitry.(b) Poisson noise—noise arising from the statistical distribution of the

recorded photons.

In general terms, detector noise is most important at very low photon levels,Poisson noise at higher levels. In a typical cooled CCD camera, the measure-ment noise, mostly readout noise from the A/D converter in the camera, isabout ten electrons (r.m.s.) per pixel. This means that if many measurementsof exactly the same number of electrons were made, the standard deviation(root mean square deviation from the mean value) would be ten. So, if exactly100 electrons had accumulated in a particular pixel in the array, there wouldbe a probability of about 70% that the measured number would be between90 and 110. The detective quantum efficiency (i.e. the fraction of incidentphotons which produce a detectable electron in the charge wells—DQE)varies between 20% and 80% depending on the wavelength and the CCDchip design. Let us assume 50%. This means that the detector noise is equiva-lent to about 20 incident photons. Poisson noise arises because the emissionand detection of photons is a random process and fluctuates according to thePoisson distribution. So if many measurements of a 'uniform' flux of photons

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Figure 4. Diagram to show contrast transfer as a function of in-plane resolution. The con-focal microscope has a better contrast transfer, especially towards the resolution limit,but this can easily be outweighed by poorer signal-to-noise characteristics.

P. J. Shaw-

were made using a perfect measurement device, then the standard deviationof these measurements would be given by the square root of the mean value.So, if the detected number of photons is 100, we can regard this as a sampleof a probability distribution whose mean is 100 and whose standard deviationis 10. Thus at a signal level of 400 photons, the Poisson noise would be 20photons—the same level as the detector noise. At this signal level we couldexpect an r.m.s. noise-to-signal ratio of around 7% (400 photons measuredwith 20 photons Poisson noise and the equivalent of 20 photons detectornoise—since the two sources of noise are independent we can add their con-tributions as the square root of the sum of their squares). With a highersignal level, the Poisson noise would be greater than the (signal-independent)detector noise.

The photomultipliers used in confocal microscopes, if they are kept cool, orat least not allowed to get warm, and if the detection circuitry is optimal, arecapable of very low detector noise levels—probably one to two counts orlower. Thus, to obtain the same 7% signal-to-noise ratio as for the CCD camerawe would only need to measure about 200 counts. (Only Poisson noise willcontribute significantly and V200/200 is about 0.07.) However, the DQE ismuch lower—perhaps only 10% or less for a typical photomultiplier—sodetecting 200 counts would require 2000 incident photons at this sort ofefficiency level.

Thus, purely in terms of image detection, the confocal microscope is clearlyworse than the best scientific CCD cameras. But another problem for the con-focal microscope comes when we consider how the photons arrive at thedetectors. In the wide-field microscope, the whole specimen is illuminated atonce and a 2D image is recorded at the camera. For typical biological fluor-escent specimens, it is common for a one second exposure to give thousandsof detected photons in the brightest pixels. On the other hand, the confocalmicroscope accumulates the image one pixel at a time, and each pixel is onlyexposed to the laser beam for a very short time (perhaps a microsecond orless). It is not easy to estimate the number of photons being measured, but ithas been estimated that in a typical specimen, the brightest pixels may onlyrepresent 10-20 photons in a single scan. Therefore to obtain the numbers ofphotons in the image that a CCD camera records in a single second exposuremay require hundreds of successive confocal scans to be accumulated. This isusually not feasible, and thus confocal images generally have a considerablyhigher noise level than wide-field images recorded by CCD cameras.

4.2 Out-of-focus lightAs has already been mentioned, the principle advantage of the confocalmicroscope is that it eliminates the unwanted out-of-focus light from measure-ment. This is entirely equivalent to the differences in the p.s.f.s between con-focal and wide-field microscopy that have been described above. However,

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the fact that the measured image can be described mathematically as the con-volution of the object with the p.s.f. means that it is in principle possible toreverse this modification by the p.s.f. This process can be accomplished byvarious computer image processing methods. A detailed description is beyondthe scope of this chapter, and the interested reader is referred elsewhere (6,10).This removal of the out-of-focus component in a wide-field image is usuallycalled deconvolution, restoration, or simply deblurring. Deconvolution methodscan also significantly improve confocal images (6). The faithfulness withwhich such a reconstruction can be achieved depends on the noise level inthe image. With no noise present, a perfect reconstruction can be carried outtrivially. But, as we have already described, all images are certain to containnoise, often at relatively high levels. In the presence of noise, image restora-tion becomes a technically difficult problem. Computer methods for handlingthis problem have been extensively developed, and suitable programs foroptical microscopy are now commercially available (see ref. 10 for a discussionof this topic and a survey of currently available software). Thus, it is possibleto collect wide-field images taking advantage of the excellent imaging charac-teristics of scientific grade CCD cameras, and using image processing to coun-teract the effects of the wide-field p.s.f. However, it should be realized thatwhile it may be possible to eliminate or reduce the out-of-focus componentfrom each focal plane by restoration methods, this unwanted light is presentin the measured image, and contributes to the noise in the measured image.The more out-of-focus light there is in the wide-field image, the more noise itadds to the image, and the smaller is the fraction of the measured photonscorresponding to the in-focus image. The confocal arrangement eliminatesthis light from measurement and, along with it, the associated noise. So themore out-of-focus light is present, the greater the benefit of eliminating itfrom measurement by the confocal arrangement. However, although the con-focal optics eliminates the out-of-focus light from detection, it does not pre-vent parts of the specimen out of the plane of focus from being illuminated bythe excitation light. Photodamage is therefore likely to be as great with con-focal as with conventional imaging, other factors being equal. Two-photonand other related methods hold out the promise of improving on this situationby restricting the specimen excitation to the centre of the focal spot.

4.3 When should confocal microscopy be used?Although the main aim of this chapter is to explain the basis of confocal imag-ing, we shall briefly discuss some of the factors that should be considered indeciding whether confocal microscopy is the most appropriate technique for aparticular imaging experiment.

Since confocal microscopes do not currently compare favourably with highgrade CCD cameras in detection sensitivity and speed of image capture, theyare not well suited to imaging very weak or photosensitive specimens. It is

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entirely possible that a confocal microscope will produce an image for a weakspecimen that is actually worse than a conventional fluorescence microscopewith an attached CCD camera. The only real advantage of a confocal micro-scope is that the out-of-focus light is eliminated. Therefore there is only anyreal point in using confocal microscopy where this advantage is important; inpractice this means for specimens with substantial thickness. But it is notalways obvious what 'substantial' means. Certainly we have obtained goodconfocal images from specimens several hundred micrometres in thickness inwhich the in-focus signal was virtually obscured by the out-of-focus light,making the conventional fluorescence image so poor as to be useless. On theother hand it is pointless using a confocal microscope to image thin micro-tome sections (a fraction of a micrometre in thickness). Better results willalmost certainly be obtained by the better image detection capabilities of agood camera on a conventional microscope.

However, most real specimens are somewhere in between these twoextremes, and it is not always easy to predict what imaging method will per-form best for a given specimen. To some extent what is used will depend onwhat facilities are available. If a very good cooled CCD camera, coupled withstate-of-the-art deconvolution software is available, and, importantly, if thereis access to expertise in using it, then it is likely that better images can be pro-duced in this way in many cases, at least for specimens which are no morethan one cell thick. There are good examples in the literature of outstanding3D reconstructions produced in this way, and several integrated imaging andrestoration packages are now commercially available.

Nevertheless the expertise necessary to achieve such good results shouldnot be underestimated. Most researchers involved in projects of which imagingis typically only one part, and who therefore cannot afford the time to learn tobe experts, will probably find it quicker to learn how to use a current confocalmicroscope adequately than a CCD/image processing workstation system.However both types of system are rapidly improving in power, affordability,and user-friendliness. The ideal situation is to have access to both types ofequipment and to use whichever provides the best imaging for the experimentin hand. There is a great deal of overlap between a confocal microscope andassociated computer and a high quality CCD microscope imaging system, inboth microscope and computer hardware, and in the software needed for imagecollection and subsequent analysis and processing. It may be hoped that oneof the various manufacturers might take the step of integrating both types ofimaging into a single instrument.

Figure 5 shows an example of the same specimen—a Drosophila embryolabelled with rhodamine-phalloidin to show the actin distribution—imaged byboth conventional fluorescence using a cooled CCD camera and by confocalfluorescence microscopy. Because this is a relatively thick specimen with agreat deal of out-of-focus light arising from other planes than the plane offocus, the difference between the two methods of imaging is dramatic.

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Figure S. Comparison of conventional, wide-field and confocal imaging. (A) An unpro-cessed conventional fluorescence image, collected using a cooled CCD camera, of aDrosophila embryo (gastrula stage) in which the actin network has been labelled withrhodamine-phalloidin. The large amount of out-of-focus light masks much of the fineimage detail. (8) A confocal image of the same specimen. The exclusion of the out-of-focus light leaves the image detail easily visible. (Specimen courtesy of Richard Warn.)The confocal microscope, CCD camera, and microscope objective were all the same as inFigure 3, In this case the fluorochrome was rhodamine, and the standard filler sets forthis fluorochrome were used. Laser excitation wavelength used was 568 nm.

5. Practical examples of specimen preparation forconfocal imaging

We conclude with two examples of specimen preparation for confocal imagingtaken from our current research into plant nuclear organisation. The purposeis not to give an exhaustive set of protocols, which are necessarily spccial-ized and depend on the biological system and type of study, but rather to illus-trate some general points and provide a link between the foregoing theoryand practical biological imaging studies. Other chapters in this volume (e.g.Chapter 6) provide more specialised protocols for specimen preparation, datacollection, and subsequent data handling.

In general terms, confocal fluorescence microscopy simply requires a fluor-escent specimen, which can be prepared in a similar way to any other lluor-escently labelled specimen. However, the ability of the confocal microscopeto produce clean optical sections from thick, thrcc-dimensionally well pre-served specimens should not be wasted. In practice, this means that some careshould be taken to preserve the three-dimensional structure of the specimenas well as possible. For living specimens, methods that keep the tissue or cellsalive and active will almost certainly preserve three-dimensional structurewell. For dead, fixed specimens the best fixative that is consistent with the

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labelling method should be used. We nearly always use formaldehyde solu-tions (which should be freshly made by dissolving solid paraformaldehyde),sometimes with a small percentage of glutaraldehyde. Electron microscopyhas shown that the bifunctional glutaraldehyde is a better fixative thanformaldehyde, but it often interferes with penetration of antibodies and otherprobes. It also causes a high autofluorescent background, which can be allevi-ated to some extent by a subsequent treatment with sodium borohydride.Fixed tissues often also need extra permeabilization to allow penetration ofprobes. In the cases of plant material, partial digestion of the cell wall withcellulase and other cell wall degrading enzymes is usually needed. Producinggood specimens usually depends on achieving a balance between preservationof the structures of interest and disrupting them so as to allow probes in tovisualize the structures.

In general, it is best to leave the physical form of tissues as unaltered as pos-sible. So in one of the protocols below we describe how to label entire rootsfrom the plant Arabidopsis. In many cases the tissue of interest is within a rel-atively large organism or organ and must be physically removed or sectionedto make labelling and imaging possible. This is the case with the large roots ofpea seedlings described in the second protocol. For these cases we routinelyuse a vibratome, which can cut quite thick sections (50-100 |xm) from living orfixed roots. Finally, the specimen should be mounted with care, so that thecoverglass does not squash the carefully preserved structure. For thin speci-mens we find that the thickness of the coating between the wells on multiwellslides is sufficient to protect the specimen from the coverglass. For thickerspecimens it may be necessary to support the coverglass. Either use nail var-nish or more coverglasses to make a platform to raise the coverglass awayfrom the specimen.

In principle any objective which can be used for conventional fluorescencecan also be used for confocal fluorescence imaging. As with all fluorescenceimaging the brightness of the image is strongly dependent on the numericalaperture (NA) of the objective and so objectives with the highest availableNAs should generally be used. As discussed above, confocal imaging is moreseriously degraded by aberrations than conventional imaging, and so it ispreferable to use high quality planapochromat objectives if possible. Howeverit should be noted that while all planapochromat objectives are suitable forfluorescence imaging with visible light (e.g. FITC, rhodamine, Texas Red,Cy3), the objectives of this type made by some manufacturers do not transmitthe UV light required for excitation of the common DNA dyes like DAPI. Infact very few available objectives transmit the commonly used UV laser wave-lengths from high power argon ion lasers efficiently (351 and 363 nm). Wenearly always use oil immersion objectives for confocal imaging, in spite ofthe spherical aberration and refractive index mismatch problems describedabove. We have obtained optically very good results from a water immersion,

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coverglass-free objective (Zeiss, X 63,1.2 NA). In fact the lower aberration inuse of this objective means that optically better images are obtained with itthan with the oil immersion objectives having higher numerical apertures (e.g.Nikon, X 60,1.4 NA, and Leitz, X 63, 1.4 NA). However, using these cover-glass-free objectives poses several problems: the specimens are not well pro-tected, and are difficult to keep; focusing the objective transfers forces to thespecimen which tend to cause it to move around and often to break up; it isdifficult to find a satisfactory water-based antifade mountant. A water im-mersion lens with a coverglass would be a better alternative. Several manu-facturers are now making these objectives, but at the moment they are veryexpensive.

Protocol 1. Whole mount immunofluorescence of Arabidopsisroots

Reagents• Bovine serum albumin (BSA) (Sigma, A- Cellulase R10 (Yakult Honsha Co. Ltd.)

7030) Pectolyase (Sigma)Nonidet P-40 (NP-40) (Fluka Chemicals Ltd.) Driselase (Sigma)PEM buffer: 50 mM Pipes (Sigma), 5 mM -y-aminopropyl tri-ethoxy-silane (APTES)EGTA (Sigma), 5 mM MgSO4, pH 6.9 with (Sigma)KOH . PBS buffer: 130 mM NaCI (Sigma), 7 mM

. Blocking solution: 3% BSA in PEM, 0.2% Na2HPO4 (Sigma), 3 mM NaH2P04 (Sigma)NP-40 pH 7.4

. 4,6-diamidino-2-phenylindole (DAPI) (Sigma): Paraformaldehyde (Sigma)1 M-Q/ml in distilled water Glutaraldehyde: EM grade, 25% solutionVectashield mounting medium (Vector Lab- (Agar Scientific Ltd.)oratories Inc.) Decon 90 (Decon Laboratories Ltd.)

Formaldehyde fixative

Add 8% (w/v) paraformaldehyde to distilled water (e.g. 2 g to 25 ml) andheat with constant stirring to 60°C. Add one drop of 1 M NaOH (for 25 ml)and continue to stir at 60°C. The cloudy suspension should clear to acolourless solution within a minute or two. Cool to room temperature, addan equal volume of 2 x concentrated PEM buffer and check the final pH. (Itis recommended that indicator paper or a dedicated pH meter is used forthis, since fixatives can damage pH electrodes, mainly by cross-linkingprotein contaminants onto the permeable plug.) If the paraformaldehydedoes not dissolve easily with this procedure (e.g. requires more NaOH tobe added) it is recommended that it should be discarded and a new batchobtained. In our experience more failures in labelling experiments can betraced to problems with fixation than to any other cause. We regularlyreplace stocks of paraformaldehyde, at intervals of six months to one year,and store it in small, separate aliquots at 4°C. Once opened, an aliquot isused or discarded after a few days.

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APTES treatment of slides

To ensure that sections adhere firmly to slides, we routinely pre-treat theslides with APTES, which we find more effective than the commonly usedtreatment with poly-L-lysine. Wash slides with ethanol or with 2% (v/v)Decon 90 or similar detergent, for a minimum of 1 h, then rinse thoroughlywith several changes of distilled water. Place slides in a freshly preparedsolution of 2% (v/v) APTES in acetone for 10 sec, then transfer to acetonealone to wash, and allow to air dry. The slides can then be stored in dust-free conditions for up to six months, but gradually lose their effectiveness.Just before they are needed, the slides are placed in a 2.5% (v/v) solutionof glutaraldehyde in PBS for at least 30 min, then rinsed in distilled water,and air dried.

Method

1. Fix roots in 4% freshly made formaldehyde in PEM, 0.2% NP-40 for 1 h.

2. Wash in PEM, 0.2% NP-40 and then in PEM alone.

3. Dry onto APTES treated multiwell slides.

4. Treat with 0.05% cellulase, 0.025% pectolyase, 1% driselase in PEMfor 10 min.

5. Wash in PEM, 0.2% NP-40 and allow to air dry.

6. Block with blocking solution for 90 min.

7. Incubate in primary antibody overnight at 4°C.

8. Wash in blocking solution for 1 h.

9. Incubate in secondary antibody for 2 h at 37°C.

10. Wash in PEM, 0.2% NP-40 for two days.

11. Counterstain with DAPI if required and mount in Vectashield or otherantifade mountant. If a coverglass is used, it should be the correctthickness for the objective lens that is intended to be used—this isusually 0.17 mm and corresponds to number 1 1/2. The imaging willgenerally be worse with any other coverglass thickness, whethergreater or less. It is important that the coverglass does not squashand flatten the specimen. If necessary, the coverglass should be sup-ported by small fragments of broken coverglass placed on the slide.The coverglass should be secured in position by nail varnish at theedge.

Figure 6 shows part of an Arabidopsis root labelled with an antibody to thespliceosomal protein U2B", which is located in the nuclei both in the inter-chromatin region and prominently in the coiled bodies.

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Figure 6. An Arabidopsis root labelled with antibody to the U2-associated spliceosomalprotein U2B". The most prominent structures fabelled by this antibody are small, spheri-cal bodies within the nuclei, ranging from one or two to several per nucleus. We haveshown that these bodies correspond to structures called coiled bodies by electron micro-scopists (because the EM ultrastructure often displays an apparent substructure of coiledfibres), A confocal section showing files of epidermal cells is shown. The files which willdevelop root hairs (e.g. the central file) contain markedly more coiled bodies than thefiles which will not make root hairs (e.g. the two files either side of the central file). Afluorescein labelled secondary antibody was used, and imaging was carried out on a Bio-Rad MRC600 confocal microscope as in Figures 3 and 5, using 488 nm excitation laserlight, and a Leitz planapo X 63 oil immersion objective (NA 1.4). Bar = 10 [sm. (Courtesyof Kurt Boudonck.)

Protocol 2. In vitro transcription on vibrato me sections of plantroots

Equipment and reagentsVibratome (e.g. Series 1000 Vibratomefrom TAAB Laboratories Equipment Ltd.)Multiwell slides: glass 8-well multitestslides from ICN Biomedicals, Inc.PB buffer: 100 mM potassium acetate(Sigma), 20 mM KCI (Sigma), 20 mM Hopes(Sigma), pH 7.4 with KOH1 mM MgCl2 (Sigma)1 mM ATP (disodium salt, from Sigma)1% (v/v)thiodiglycol (Sigma)2 ng/ml aprotinin (Sigma)Triton X-100 (Sigma)

0.5 mM PMSF (phenylmethylsulfonyl fluo-ride, Sigma): make up as 100 mM stocksolution in ethanol, store at -20oC, and addto buffer just before useTranscription mix: 50-500 jJV! CTP (sodiumsalt, Pharmacia), 50-500 (iM GTP (sodiumsalt, Pharmacia), 25-250 iiM BrUTP (sodiumsalt, Sigma), 125 ̂ M MgCl2, 100 U/ml RNAGuard (Pharmacia), made up in PBTBS: 25 mM Tris-HCI pH 7,4 (Sigma), 140mM MaCI, 3 mM KCIHexylene glycol (2-methyl 2,4-pentane-diol, Sigma)

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Method

1. Excise the first 3-4 rr»M of a freshly germinated root tip and mount onthe vibratome. Cut sections (40-50 \i.m) in PB containing 1 M hexyleneglycol and transfer to tissue handling device (11).

2. Incubate in 0.05% Triton X-100 in PB for 1 min, then wash in PB alone,three times over ~ 30 sec.

3. Incubate in transcription mix for 5 min or other times, then washagain in PB alone, three times over ~ 30 sec. The lower concentrationof NTPs given is the minimum that will support transcription in ourexperience, the higher concentration routinely gives high levels oftranscription.

4. Fix in 4% freshly made formaldehyde in PEM (see Protocol 1) for 1 h.

5. Wash in TBS for 10 min, then H20 for 10 min.

6. Remove from tissue handling device and allow to air dry onto APTEScoated multiwell slides (see Protocol 1).

7. Treat sections with 2% cellulase (Onozuka R10) in TBS for 1 h, thenwash in TBS, three changes over 15 min.

8. Incubate for 1 h with primary antibody (mouse anti-BrdU; Boehringer),diluted 1:20 in PBS, 3% BSA.

9. Wash thoroughly with TBS, then incubate for 1 h with secondaryfluorescent antibody (Cy3-conjugated anti-mouse; Jackson Immuno-research).

10. Mount in Vectashield or other antifade mountant.

Figure 7 shows a section of pea root labelled in this way to show the sites ofRNA transcription. The nucleoli in the centre of the nuclei are most stronglylabelled and correspond to incorporation by RNA polymerase I, with manydispersed transcription sites corresponding to RNA polymerase II and IIIthroughout the rest of the nucleus.

Protocol 3. Collection of confocal images

This is not really a protocol so much as a set of procedures that should beused in optimizing the parameters for collection of confocal images. Inprinciple it should apply to any confocal microscope, but the details will dif-fer on different machines. The settings described in steps 1-4 all interactwith each other, and must often be iterated to produce the best image.What constitutes the 'best' image also depends on the imaging experimentbeing undertaken. Ideally, for multiprobe imaging of several different

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fluorochromes, steps 1-4 should be optimized for each fluorescent probein turn, although this is not always possible.

Method

1. Optimize the laser illumination intensity. This depends on the specimenand the fluorescent labelling. In general the highest intensity that willnot cause problems should be used. Problems from too much illumin-ation light include fading of the fluorochrome and photodamage toliving cells. It should be remembered that the very high laser lightlevels used can easily cause a complete population inversion of thefluorescent molecules to the excited state; any further excitation willonly cause photodamage. Often it is better to use a lower illuminatinglight level, and average the image over a longer time.

2. Optimize the detector pin-hole diameter. The smaller the pin-hole, thebetter the resolution—primarily the better the exclusion of out-of-focus light, but the less light is detected and the poorer the statisticalproperties of the resulting image. It is often better to open up the pin-hole for a weak specimen, and compromise on the confocality of theimage.

3. Optimize the setting of the photomultiplier amplifier circuitry, usuallyprovided as a gain and black-level setting. In general this should be setso that maximum and minimum detected signals in the image corre-spond to the highest and lowest digitized level respectively—i.e. usually255 and 0 for an 8-bit analogue to digital conversion. It is often usefulto arrange for these values in the image to be flagged as different dis-play colours, and the computer software will often provide a speciallook-up table (LUT) for this purpose. If the minimum and maximumare set too low and high, then the image grey levels will not span theavailable range and will be compressed into a smaller grey level rangethan necessary. This could result in the loss of image information. Ifthe minimum is set too high or the maximum is set too low, this willcause artefactual thresholding of the image at the set minimum ormaximum, and again may result in the loss of image information orthe introduction of artefacts.

4. Optimize the image averaging. Single image scans usually show poorimage statistics—i.e. high levels of image noise—simply because ofPoisson noise given the low numbers of photons detected in a singlescan. The solution is to accumulate many scans of the image, or to scanat a slower rate, or both. The image signal-to-noise ratio increases asthe square root of the number of scans accumulated. The disadvan-tage to accumulating many scans is the time taken and the increasedlight dosage, causing fluorochrome fading, photodamage, etc. In thecase of living cells, dynamic changes may occur and be blurred by

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long accumulation series. The requirement for dynamic informationand good image signal-to-noise must be balanced against each other.

5. Set up the other data collection parameters, such as for focal series(z series), time series, etc., and collect the image data.

Figure 7. A pea root section labelled by BrUTP incorporation. The strongest labelling isfrom incorporation by RNA polymerase I into pre-rRNA in the nucleolus, but all cells alsoshow labelling in the nucleoplasm in many disperses foci, which must be due to incorpo-ration by RNA polymerases II and III. The confocal microscope used was an MRC600 (asin the previous figures). An argon/krypton mixed gas laser was used In the confocalmicroscope, and an excitation wavelength of 568 nm was used to excite the fluo-rochrome (Cy3). Bar = 10 um, (Courtesy of Alison Beven.)

References1. Stelzer, E. H. K.. Lindek, S., Albrecht, S., Pick. R., Ritter. G., Salmon, N. J., et al.

(1995),J, Microsc.,179, I.2. Pawley, J. B. (ed.) (1995). Handbook of biological confocal microscopy. Plenum

Press, New York and London.3. Castleman, K. R. (1979). Digital image processing. Prentice-Hall,4. Petran, M., Hadravsky, M., Egger. M. D., and Galambos, R. (1968), J. Opt. Soc.

Am.,58.66l.5. Juskaitis, R., Wilson, T.,Neil, M. A. A., and Kozubek, M. (1996). Nature, 383, 804.

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6. Shaw, P. J. (1995). In Handbook of biological confocal microscopy (ed. J. B.Pawley), p. 373. Plenum Press, New York and London.

7. Inoue, S. (1995). In Handbook of biological confocal microscopy (ed. J. B.Pawley), p. 1. Plenum Press, New York and London.

8. Pawley, J. B. (1995). In Handbook of biological confocal microscopy (ed. J. B.Pawley), p. 19. Plenum Press, New York and London.

9. Sheppard, C. J. R., Gan, X., Gu, M., and Roy, M. (1995). In Handbook of biologi-cal confocal microscopy (ed. J. B. Pawley), p. 363. Plenum Press, New York andLondon.

10. Shaw, P. J. (1997). In Cell biology: a laboratory handbook (ed. J. Celis), Vol. 3,p. 206. Academic Press, New York and London.

11. Wells, B. (1985). Micron Microsc. Acta, 16,49.

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Video microscopyDIETER G. WEISS, WILLI MAILE, ROBERT A. WICK, and

WALTER STEFFEN

1. Video microscopy and the equipment required1.1 IntroductionA new quality of microscopy, called video microscopy, emerges, if one observesthe specimen, instead of with the human eye, with a video camera connected tovideo processing equipment working at real time. Video microscopy is, there-fore, much more than just adding a camera and monitor to the microscope toshare the images with a larger audience. More recently, electronic devicesother than video cameras, such as high sensitivity charge coupled device(CCD) cameras and scanning light detector systems for confocal microscopyhave been added to microscopes. The three fields (i) video-enhanced contrastmicroscopy for highest resolution work, (ii) video-intensified microscopy forlow light applications, and (iii) electronic scanning microscopy for confocalmicroscopy and 3D imaging differ in the type of device generating the elec-tronic image, but all three use basically the same types of analogue and digitalimage processors. While all these techniques are generally defined as elec-tronic light microscopy, this chapter, video microscopy, deals with the first twotechniques that involve CCD and video cameras as imaging devices.

Video microscopy has produced a revolution in light microscopy of biologi-cal samples equivalent to that of the development of the immunofluorescencetechnique. It has once more made the traditional light microscope a powerfultool for those working on dynamic aspects of small biological systems, forexample biochemists, molecular and cell biologists. It has given further resolv-ing power to the light microscope enabling the observation of particles whichbridge the size range between those normally studied by electron microscopyand those which are already well known to light microscopists as a whole, withthe added advantage in that specimens can be examined alive. As well asallowing small particles to be resolved, the technique has the capacity to cleanup the image, so allowing greater visibility. Also, changes of such parametersas amounts, concentrations, transport, or metabolism of specific molecules inboth tune and space can be quantitatively determined.

The improvement in resolution is achieved because a microscope equipped

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Dieter G. Weiss, Willi Maile, Robert A. Wick, and Walter Steffen

Figure 1. A possible circuit diagram for video microscopy containing equipment forimage acquisition, analogue and digital image processing, image display and recording.At least one B/W monitor is required, but one monitor showing the unprocessed liveimage is recommended for comparison and focusing. ALU = arithmetic logic unit; A/DC= analogue/digital converter; D/AC = digital/analogue converter; LUT = look-up table;ODR = optical disk recorder; RGB = red green blue; Sync = synchronization signal;VTR = video tape recorder.

with an electronic camera, analogue and digital image processors, and electronicdisplay, recording, and printing devices (Figure 1) is able to detect differencesin intensity which are far smaller than those detectable in a conventionalmicroscope with the human eye. For the same reason, weakly self-luminousobjects can be found and characterized as images.

The greatest improvements in microscope imaging are only possible on thebasis of very precise optical microscopy arising from a thorough knowledge ofthe principles of light microscopy, for example of Kohler illumination andconjugate optical planes. However, it should also be said that, on occasion, theless skilled microscopist might be able to considerably improve the quality ofthe images when using video microscopy. Another major advantage of the tech-nique is that it provides easy recording of information on video tape or diskfor later quantitative analysis. Finally, the ability to store and post-processimages with PC-based software packages enables microscopists to re-examinetheir previously obtained data, compare them with more recently producedimages, and optimize and finally arrange them for publication.

In video microscopy we deal with images that contain the intensity informa-tion encoded in either analogue or digital form:

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3: Video microscopy

(a) Analogue: the brightness at each point of the optical microscope image isconverted into a voltage signal by the camera. The analogue signal isa continuous signal where 0.4 V represents black and 1 V white. It isinterrupted by synchronization signals defining the end of lines and fields.Normally, one frame consists of 576 visible lines (European standard,CCIR) divided into two fields (half frames) containing either all evennumbered or all odd numbered lines ('interlaced mode').

(b) Digital: by the use of an analogue-to-digital converter (A/DC) the con-tinuous voltage signal is converted into discrete numbers which areassigned to an array of picture elements (pixels). A common format is768 X 576, i.e. 768 pixels per line and 576 lines. If an 8-bit conversion isused, one obtains images with 256 grey levels where 0 represents blackand 255 white.

In analogue image processing contrast can be amplified electronically up toseveral thousand-fold. In digital image processing contrast is enhanced numeric-ally. The upper limit of useful digital enhancement is only about threefold;however, image quality can be further improved considerably by a large num-ber of additional algorithms.

Video microscopy, as dealt with here, involves the generation or improve-ment of microscopic images in three basic ways (Sections 1.1.1-1.1.3).

1.1.1 Video enhancementVideo enhancement is the procedure of increasing contrast electronically inlow contrast or 'flat' images. This process not only clarifies images containingdetails visible to the eye, but renders visible structures 5-20 times smaller thancould be detected by vision alone or in photomicrographs (Figure 2).

Video-enhanced contrast (VEC) microscopy was developed in the laborato-ries of S. Inoue (1, 2) and R. D. Allen (3-5). Allen et al. (3, 4) found that theuse of VEC microscopy with polarized light methods allowed the introductionof additional bias retardation, which, after offset adjustment and analogueenhancement, permitted much better visualization of minute objects. AVEC(Allen video-enhanced contrast) microscopy is the term used to describe thistechnique.

1.1.2 Video intensificationVideo intensification is the procedure for making visible low light level objectsand scenes generating too few photons to be seen by the naked eye (Figure 2).Video-intensified microscopy (VIM) amplifies low light images so that veryweak fluorescence and luminescence can be visualized (6,7). This is especiallyimportant in biology because living specimens benefit from the sparing appli-cation of potentially hazardous vital dyes or excessive illumination. VIM andVEC microscopy differ mainly with respect to the type of camera used; i.e.

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Figure 2. Video microscopy works beyond the former limits of light microscopy. Newapplications are opened (dotted areas) both in low light situations (VIM) and when work-ing with very small objects (VEC microscopy). All borders are meant to be approximate.

low light level cameras for VIM and high spatial resolution cameras for VECmicroscopy.

1.1.3 Digital image processingMicroscopic images that have been picked up by a video camera can be con-verted to a digital signal allowing digital image processing to be performed.Image processing can be used to reduce image noise by digital filtering oraveraging, to subtract undesired background patterns, to further enhance con-trast digitally, or to perform measurements in the image (e.g. intensity, size,speed, or form of objects). Since the development of procedures for noisereduction and contrast enhancement in real time, that is at video frequency,the microscopist is able to generate electronically optimized pictures whileworking at the microscope. The procedures to be applied here are generallyvery similar to those used with other types of electronic imaging with videocameras or photomultiplier tubes, such as confocal microscopy (see Chapter 2)or scanning electron microscopy (SEM).

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1.2 General strategies of electronic image improvement1.2.1 For photon-limited situationsIn photon-limited situations, especially with fluorescent, luminescent, andmost dark-field specimens, video intensification is required. VIM techniquesrequire the use of low light level cameras which, unfortunately, are usually nothigh resolution cameras. It may be possible, however, to visualize highly fluor-escent objects considerably below the limit of resolution if they are wellseparated from one another (e.g. a diluted suspension of fluorescent actin fila-ments). The procedure in low light level microscopy would usually requirethe full range of video-microscopic techniques, that is the use of a video-intensification camera, analogue enhancement, and a variety of digital imageprocessing steps such as real time background subtraction (to compensate theuneven sensitivity of some VIM cameras), averaging to reduce noise, and digitalenhancement (see Section 3). VIM images with their smooth transitions(low spatial frequencies) are usually well suited for digital image analysis ofintensities and intensity changes (see Section 4.2).

1.2.2 For high image fidelity and detailIf high image fidelity and detail are desired and the smallest objects of interestare larger than the limit of resolution of the microscope (~ 200 nm), then highresolution cameras are appropriate. They require a fair amount of light but canbe used with differential interference contrast (DIC) and all brighter tech-niques. In such a case we would use VEC microscopy image improvement, butmoderate analogue enhancement may suffice. If uneven shading occurs in theimage then an analogue shading correction is usually sufficient at this level ofmagnification. Background correction by digital processing, or digital en-hancement is usually unnecessary. Typical applications include observations ofwhole cells for studies of cell form, cell division, or movement of large organ-elles. Also, studies at low or intermediate magnifications of small organisms,embryos, or developing eggs, making use of the optical sectioning capacity ofDIC microscopy, will benefit considerably.

1.2.3 For visualizing the smallest objects possibleIf it is desired to visualize the smallest objects possible, one would also useVEC microscopy, preferentially employing DIC or anaxial illumination tech-niques. Visualization of microtubules (25 nm in diameter) or vesicles withdiameters of 50 nm or less can be achieved (8, 9). For this purpose we wouldneed the following functions of electronic image improvement: high analogueenhancement, high performance polarized light microscopy [DIC or POLmicroscopy according to Allen (3, 4) or Inoue (1)], and digital image process-ing including real time background subtraction, and digital enhancement. Ifthe resulting image is noisy, real time averaging over two or four frames orreal time digital filtering might be employed.

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For most users VEC and VIM microscopy are complementary since theformer reveals the intracellular structures while the latter, with the use offluorescent tags such as dyes or fluorescent antibodies, is needed to determinethe identity of the objects depicted.

For the microscopist it is most important that the equipment operatesrapidly, that is the processor must operate sufficiently fast to display changesin the image in real time.

1.3 The different video-microscopic techniques1.3.1 Video-intensified microscopyVideo-intensified microscopy has greatly extended the capabilities of the lightmicroscope (Figure 2) and has provided the technical vehicle for the develop-ment of several important new techniques. VIM has made possible the observ-ation and recording of images too weak to be seen by direct viewing or filmrecording. Furthermore, it has provided a mechanism to study living cells forextended periods without disrupting normal metabolic activity or bleachingphotosensitive molecules.

Examples of naturally occurring low light phenomena, such as autofluor-escence or bioluminescence, are widespread. Moreover, the use of exogenousluminescent and fluorescent molecules (e.g. fluorescent antibodies or genetic-ally modified proteins tagged with green fluorescent protein) as probes ofcellular structure and function has become an important tool in almost allareas of biological research. The application of VIM in these research areashas been reviewed (6,10,11).

Video-intensified microscopy is particularly useful in the following situations:

(a) Where the total number of photons available for imaging is limited by thenature of the event, as in bioluminescence or in fluorescence where thenumber of labelled molecules is small.

(b) Where low intensity illumination is required to avoid interfering with thebiological process(es) under investigation or to avoid phototoxic effects.

(c) Where there are rapid changes and the amount of light available in thetime interval studied are small.

(d) Where the long exposure times necessary for photography could preventthe recording of dynamic processes, e.g. fluorescence from cytoplasmiccomponents in living cells.

(e) Where fluorescence excitation needs to be minimized to reduce photo-bleaching.

(f) Where labelling is intentionally limited to avoid biological interferencefrom, for example, toxic dyes.

While the procedures of analogue contrast enhancement (see Section 1.3.2)may not be required routinely, digital image processing is still very helpful,especially for noise reduction.

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1.3.2 Video contrast enhancementVideo-enhanced contrast (VEC) microscopy, enables one to increase contrastand magnification to an extent that positions and movements of biologicalobjects as small as 15-20 nm can be analysed in the living state. VEC micro-scopy is especially useful to the cell biologist, biochemist, and the molecularbiologist because:

(a) Objects beyond the limit of resolution of conventional light microscopycan be visualized (e.g. microtubules with a diameter of 25 nm).

(b) It enables one to visualize cell organelles and supramolecular aggregatesin living cells.

(c) Under certain circumstances, it allows quantitative measurements ofamount, concentration, transport activity, or metabolism of specificmolecules.

1.3.3 Analogue contrast enhancementThe introduction of analogue enhancement resulted in the remarkable break-through which led to a new level of performance of light microscopy. Digitalenhancement, as discussed later, is often useful as an addition but it must beemphasized that it cannot replace analogue enhancement. An important basicrule is that only optimized analogue signals should be digitized and processedfurther.

Understanding the image manipulations required for analogue contrastenhancement is complicated and a basic understanding of straylight, contrast,and resolution is required. A short review of each of these aspects is thereforeincluded here.

i. StraylightLight distributed evenly over the image and not contributing to image detailis called straylight. Some of its origins are summarized in Table 1. In manycases straylight prevents the use of otherwise optimal settings of the micro-scope. For example, the resolution achievable is often sacrificed by closingthe condenser diaphragm, thus reducing the numerical aperture (NA), inorder to avoid too bright an illumination. When polarized light is used thereis usually an annoying contribution of unpolarized straylight, even at thehighest extinction settings of the polarizers or prisms. In the video image theeffect of such straylight can be removed electronically by applying a negativeDC voltage to the video signal, called the offset or pedestal voltage. By apply-ing suitable gain to the camera signal the contrast is enhanced, by using avariable offset the camera signal is shifted to the appropriate region of greylevels (brightness) for best visibility on the video screen (Figures 3b and 4).In Figure 3 the improvements achievable by image processing are shown foreach step of the technique.

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Table 1. The various sources of straylight which may beremoved by applying offset

Bright-field microscopy

Excessive condenser apertureUncoated lens surfacesReflected light from tube inner surfaces

Polarized light and interference microscopy

Optical rotation at lens surfacesStrain birefringence in lensesLight scatter due to dust, lens cement, etc.Surface imperfections in lensesDefects (holes) in polarizing filtersSubmaximal compensation

Fluorescence microscopy

Autofluorescence of any material in the light pathNon-specific localization of fluorochromesIncomplete removal of excitation lightBleed-through of one fluorescence in double labellingexperiments due to imperfect filters

21. ContrastThe brightness at each point of the optical microscope image is converted intoa voltage signal by the television camera. Contrast (C) for the eye is perceivedapproximately as the absolute value of the difference between the intensity(or brightness) of the background (IB) and that of the specimen (IS), dividedby the intensity of the background:

Contrast can be amplified, within a factor of 100 or more by the gain appliedto the camera signal, provided the proper offset setting is used.

111. Contrast manipulationThe manipulation of contrast may be applied to the images in any mode ofoptical microscopy. With polarized light, considerable additional contrast canbe achieved by adjusting the compensator to a higher bias retardation (AVECmicroscopy, see Sections 2.1 and 2.3). The resulting images are usually ofinadequate visual contrast because the denominator of Equation 1 is too highdue to excessive straylight (see Sections 2.1, 2.3.4, and 2.3.5). However, in theanalogue image, the offset voltage applied to the video signal acts in a manneranalogous to a 'negative brightness or intensity' (Iv), which is added to the

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Figure3. Processing of poor contrast image. The specimen, an unstained electron micro-scope (EM) thin section of striated muscle, was viewed by differential interference con-trast microscopy. Video microscopy was intentionally carried out prior to proper cleaningof the optics to demonstrate the procedure in the presence of unusually heavy mottle,(a) In-focus, not enhanced, (b) In-focus, analogue-enhanced, (c) Out-of-focus, with mottle,(d) Out-of-focus, mottle subtracted, (e) In-focus, mottle subtracted, (f) Digitally enhanced.Microscope, Zeiss IM 405, Plan Neofluar, x 63, NA 1,4, x 16 eyepiece, 63 mm cameralens, processor ARGUS, Hamamatsu Photonics. Frame width = 42 um.

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denominator. Video contrast (Cv) is then expressed as Equation 2, where A isthe electronic amplification obtained by setting the gain.

Once the straylight has been compensated for by the offset (pedestal) voltage,the analogue gain of the camera can be adjusted once more to utilize the fullrange of grey scales in the unprocessed image.

iv. Contrast enhancement and resolution of objectsThe gain in resolution is about twofold. Using the best lenses, where aberrationsare negligible, resolution, i.e. the smallest distinguishable distance betweentwo points, is only limited by the size of the spreading of the image points dueto diffraction, that is by the size of the Airy disk. One reason why resolutioncan be somewhat increased by contrast enhancement is that the Rayleighcriterion of resolution, that is defined as a 15% drop between the two peaksthat the eye can perceive (Figure 4a), is replaced by the Sparrow criterion (12)(Figure 4c and d) which the video camera can detect. This is applicable toelectronic images because they can be enhanced, so that even a slight troughin the intensity distribution of the two unprocessed images (Figure 4c) can beenhanced to give good separation (Figure 4d).

v. Contrast enhancement and visualization of objectsObjects smaller than the limit of resolution create a blurred image or diffrac-tion pattern known as the Airy pattern whose amplitude (intensity) is very

Figure 4. Improvement of resolution by VEC microscopy. The diffraction pattern (Airypattern) of a very small object is characterized by a central zero order maximum andsmaller maxima of first, second, and higher orders, (a) The overlapping images of twoclosely adjacent objects (pin-holes) with their summed intensity distribution (dashed) areshown. The two objects are resolved according to Rayleigh's criterion since the centraldepression is sufficiently deep to be perceivable, (b) A much improved image is obtainedby redefining the low intensity (black) end at the position indicated by the horizontal lineby applying offset, and subsequently amplifying the signal by applying gain, (c) Thesame objects are somewhat closer so that they are not resolved according to Rayleigh'scriterion, (d) However, if contrast is enhanced as for (b), even in this situation an imagecan be obtained which shows the two objects separated. Sparrow's limit of resolution isreached when there is no trough between the two peaks.

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Figure 5. Improvement of visualization of subresolution size objects by VEC microscopy.Positive objects (slits) that are larger than (a), equal in size (b), and much smaller (c) thanthe limit of resolution are imaged by transmitted light (arrows) using an ideal diffraction-limited optical system represented schematically by a single lens. The top panels showthe resulting intensity distributions across the images (diffraction disks, Airy disks).Before digitization, this corresponds to the voltage of the analogue video signal, i.e.brightness, along a video scan line. The subresolution size object (c) yields a very lowcontrast 'image' which cannot normally be distinguished from surrounding noise andtherefore remains indiscernible by eye. However, its contrast can be enhanced by apply-ing offset and gain, i.e. applying a negative DC voltage of a magnitude indicated by thedashed line and subsequent electronic amplification. This results in the definition of anew black level (intensity zero) and a higher signal, as seen in (d). As a result of such ana-logue contrast enhancement, objects much smaller than the limit of resolution (c) can beclearly visualized (d). However, their real size and shape cannot necessarily be inferredfrom the size or shape of their 'images', such as image (d) of object (c), which is inflatedby diffraction to be equal in size to image (b). (Reproduced with permission from ref. 13.)

small, but their size cannot be reduced further (Figure 5c). Usually the Airyrings around larger objects are negligible. If, however, the size of the objectbecomes smaller than the wavelength of the light used, the diffraction patternmay be larger than the object. In the best lenses aberrations have been madenegligible and image quality is only limited by the spreading of the Airypattern due to diffraction. By applying video enhancement such normallyinvisibly weak Airy patterns can be visualized. However, if two objects areseparated by a distance less than the limit of resolution their diffraction imageswill merge. Hence, by using contrast enhancement such objects can be visual-ized (Figure 5d) although they can not be resolved (Figure 4). UsingNomarski-DIC and VEC microscopy, biological structures of 15-20 nm canbe visualized, while inorganic materials, such as colloidal gold particles, canbe visualized down to sizes of 5 nm and less (see Chapter 12). In DIC micro-scopy the shadowcast diffraction patterns cancel out for many very smallobjects located at distances less than the limit of resolution and thus remain

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invisible. This situation is met in many types of small cells and nerve endingsdensely packed with organelles.

vi. Advantages of analogue contrast enhancement

(a) Straylight is removable in the video situation by offset (IV).(b) The practical resolution is increased by a factor of about two over con-

ventional microscopy. This is partly because it becomes now possible touse the maximal condenser numerical aperture since the resulting exces-sive image brightness due to Straylight can be suppressed electronicallywith offset, and partly because Rayleigh's criterion of the limit of resolu-tion is replaced by Sparrow's criterion.

(c) The gain in contrast is sufficient to visualize structures in living cellsthat are about one order of magnitude smaller than could be detectedpreviously under the same conditions (Figure 2).

(d) The AVEC conditions reduce the diffraction anomalies, caused by de-polarization at lens surfaces or by residual strain birefringence in thelenses, that produce spurious detail and contrast in conventional polar-ized light-based techniques (see Section 2.1).

vii. Limitations of analogue contrast enhancement

(a) Electronic noise is amplified along with the video signal in the enhance-ment process and may have to be subsequently reduced (see Section 2.3).

(b) If the optical system (including the slide and coverglass) contains dust,dirt, or manufacturing imperfections, these will create a fixed pattern ofmottle that is enhanced along with the image. This can only be removedby digital processing (Figure 3e) (see Section 2.3).

(c) If the illuminating system is poorly designed or incorrectly adjusted forKohler illumination, the field may be unevenly illuminated, since slightunevenness of the illumination will also be enhanced considerably. There-fore, the requirement of even illumination is much more stringent forVEC microscopy than for conventional photomicroscopy. Note, however,that within certain limits, uneven illumination can also be treated asfixed pattern noise and removed by digital subtraction (Figure 6) (seeSection 2.3).

1.3.4 Digital image processingDigital image processing such as digital filtering, background subtraction, oraveraging may be performed once the video image has been converted into adigital signal. Many of the digital image processing routines were availablelong before their value was recognized by microscopists (5, 14). With therapid development of faster computers many of these routines became avail-able at video frequency. The principles of digital image processing and someof the procedures to be employed will be described below. In VIM and VEC

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Figure 6. Correction of uneven illumination (shading) in analogue-enhanced images bybackground subtraction. If after fixing potential flaws in the optics (see Section 2.3.2) andpossibly correcting the shading in analogue mode (see Section 1.3.3) an image as in (a)appears, that shows an annoying hot spot after analogue enhancement (b), subtraction ofa specimen-free mottle image (c) will result in an evenly illuminated image (d). Thissequence also demonstrates the usefulness of a calibratable scale bar, the timer bar(months to 1/100 sec), and the intensity measurement along a line (see Section 4.2).Specimen is the test diatom Amphipleura pellucida with a known line spacing of 250 nm.Microscope, Zeiss Axiophot, Planapochromat x100, NA 1.25, Hamamatsu PhotonicsMicroscopy System,

microscopy they arc used for rapid pre-processing, that is improvement of theimages prior to their storage on tape or disk. It should be noted, however, thatindividual frames, once stored, can also he subjected to subsequent digitalprocessing and image analysis employing essentially the techniques and imageprocessors discussed in Chapter 8.

Following analogue contrast enhancement, the analogue TV signal (a tem-poral pattern of voltage changes), is digitized so that it can he manipulated bythe arithmetic logic unit (ALU) in an image processor. In the most suitableprocessors, the instructions necessary to carry out a number of different

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arithmetic manipulations are controlled by firmware or by software packagesso that an operator can easily process images combining the most suitableprocedures for his/her particular operation. The operations themselves arecarried out in 'real time', that is repetitively during the intervals between con-secutive frames (i.e. 25 or 30 times per second depending on the TV standardused).

During digitization the image is subdivided into pixels assuming one out of256 (8-bit) grey levels. Depending on the complexity of the operations to beperformed, one or more frame memories (usually 512 X 512 or 768 X 576 pixels)are required. If averaging or other multiframe procedures are to be used theALU must be capable of 16-bit operations to avoid truncation errors. Thespatial resolution is much poorer than that of a photograph that may have aresolution of approx. 5000 line pairs for 35 mm film. In principle, comparableresolution may be achieved by image analysis systems capable of handlingimages having a spatial resolution of 4096 X 4096 pixels, but these, if existing,would be extremely expensive. Therefore, additional optical magnificationmust be applied for highest resolution video microscopy (see Sections 1.5and 1.6).

i. Rolling average or jumping averageThe rolling average function computes the average of the last incoming imageand the previously stored average. This procedure results in an exponentiallyweighted average with the most recent frames dominating (recursive filter-ing). In jumping average mode a time average is computed from a pre-definednumber of frames and this is displayed for the duration of the accumulation ofthe next set of frames. Both modes diminish electronic noise in the videosignal by the square root of the number of frames averaged. The formersmears and de-emphasizes any motion present, while the latter accentuatesslow motion. Both are generally used for VIM where photon fluctuations andelectronic noise from the imaging device often present severe problems andthey are advisable for VEC microscopy when high enhancement is used, thatis when electronic noise becomes annoying. The Kalman filter (15) representsanother, often better way to produce a continuously displayed image withreduced noise.

ii. Mottle subtractionPatterns of image imperfections (mottle) remain in the analogue image whenthe specimen is defocused or moved out of the field of view (Figure 3c). Con-sequently, mottle can be stored in a video frame memory and then subtractedfrom each frame of the incoming video signal (Figure 3d). This operation(mottle or background subtraction) results in a 'clean' image lacking mottle(Figure 3e). The same operation also eliminates inhomogeneities in back-ground brightness (Figure 6), if their contrast does not exceed the range ofgrey levels, which can be handled by the processor (usually 256).

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Hi. Digital contrast enhancementThe analogue-enhanced, mottle -subtracted image may not have sufficient con-trast. In this case the image can be enhanced further digitally, e.g. by stretch-ing the histogram of grey levels (Figure 3f). The procedure is analogous toanalogue enhancement but the selection is made digitally by choosing thatrestricted region of the grey levels containing the image information andexpanding it to stretch the entire distance from black to white, that is 256 greylevels. This is done by assigning new grey levels to the original ones throughthe use of an output look-up table (LUT). Please note that analogue enhance-ment cannot be replaced by digital enhancement.

iv. Enhancement of motion by sequential subtractionThe analogue-enhanced image can be subjected to sequential subtraction inorder to observe and detect only moving elements. This is done by freezing areference image without taking the specimen out of the field or out-of-focusand then subtracting it from all incoming frames. Subtraction of a (stored)image from very similar subsequent (i.e. the incoming live) images results inblank images in which only moving elements cause image differences and somake their presence known (Figure 7). This is an extremely sensitive means of

Figure7. Selective visualization of moving objects by sequential subtraction, (a) Whenthe reference image taken at time 29:40 sec is subtracted from the incoming video imageat time 29:50 sec, i.e. 0.1 sec or five frames later, almost no contrast is visible becauseall objects remained close to their original location, (b) 2 sec later (31:50 sec) the movingorganelles became visible. Each organelle is depicted twice, once appearing as a depres-sion and once in positive contrast. The depression marks the organelle's location atcommencement, i.e. the locus where the organelle is now missing, while on the videoscreen the actually moving objects become visible in positive contrast. In this sequencein a bundle of pike olfactory nerve axons of 0.25 um diameter the movement of mito-chondria (elongate) and lysosome-like organelles (round) is observed. Most organellesexcept one mitochondrion near the centre can be seen moving to the lower right. Micro-scope, Polyvar 1, Leica/Reichert/Cambridge Instruments, Planapochromat x 100, NA1.32, Hamamatsu Photonics Microscopy System, Scale bar = 2,7 (um.

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motion detection, but only works satisfactorily with a very stable microscopestand under good temperature control. Any drift in focus or pressure appliedto the stage or microscope body may result in a distorted image. This modegives both the position of the moving object at time zero (frozen and in nega-tive contrast, i.e. a 'missing object') and the live position of the moving object(Figure 7). Distance measurements for velocity calculations can be very con-veniently obtained by this technique.

v. Interval subtractionThis method is an alternative mode of sequential subtraction that is pro-grammed to refresh after predetermined intervals, i.e. a new 'background'image to be subtracted from incoming video images is automatically chosenand stored after a certain, pre-selectable number of frames.

vi. Pseudocolour displayThis process allows the user to systematically or arbitrarily assign colours tovarious grey levels. This can be very helpful in discerning patterns or smalldifferences in intensity since the human eye distinguishes only about 70-90grey shades but a multiple of colour shades.

Additional digital functions which prove particularly useful for VIMinclude the following:

vii. Frame (or beam) blankingThis is another method for improving signal-to-noise ratio (S/N) during imageacquisition. Here, the charge pattern on the faceplate of the camera is allowedto accumulate for an extended period of time rather than being 'read out'every 1/25 or 1/30 of a second (video rate). This greatly reduces the amount ofreadout noise. For example, a typical 16-frame averaging uses 16 images, eachcontaining a certain percentage of readout noise. If, however, the image isallowed to integrate on the camera target for a period equivalent to 16 framesbefore being read out the amount of readout noise is reduced to l/16th.

viii. Spatial filteringBy applying various convolution operations the image can be 'filtered' in thespatial domain. This process allows for suppression of noise or the accentuat-ion of high frequency information as in edge detection or image sharpening(see Chapter 8 for details).

ix. Arithmetic operationsThis permits the application of the basic arithmetic operations to a singleimage or between multiple images. Ratio imaging, where one image is dividedby another, is a typical example (see Section 3.3.2).

x. Image superimpositionThe ability to superimpose one image upon another is very useful in manyapplications. Examples include combining a pseudocoloured fluorescence

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image with the corresponding black and white transmitted light image or com-bining two images, each of which represents a different fluorescent label, toevaluate their relationship to one another.

It is clear that a multitude of additional procedures to accentuate specificfeatures have been developed and are now accessible with programmabledigital image processors (see ref. 14 for overview).

1.4 Electronic equipment for video microscopyOnce you have determined the functions which will be essential for yourresearch (see Section 1.2) you need to select the appropriate type of video-microscopical equipment. A generalized scheme is set out in Figure 1. Cam-eras and image processors are discussed in this section, while comments onsuitable ancillary equipment such as recorders and monitors will be found inSection 5. VIM and VEC microscopy differ mainly in the types of camerasappropriate for each technique. A publication by the Centre for LightMicroscopy and Imaging and Biotechnology (at Carnegie Mellon University,Pittsburgh, PA, USA) will provide additional useful information for choosingthe appropriate camera (16).

1.4.1 Cameras for video-enhanced microscopyFor VEC microscopy historically exclusively high resolution vidicon cameraswere used, mainly of the Chalnicon, Newvicon, or Pasecon type. If specialspectral requirements such as high sensitivity in the infrared, red, or UVrange, or extremely low lag are desired, suitable cameras may be selectedfrom various manufacturers, such as Hamamatsu Photonics, DAGE-MTI,Inc., COHU, Inc., and others. Over the last decade CCD (charge coupleddevice) cameras have improved substantially in terms of resolution and sensi-tivity. Black and white (B/W) tube cameras with 3/4 or 1 inch tubes are stilloften used, if images are to be processed in analogue mode. CCD cameraswith 1/3 or 1/2 inch chip size are progressively replacing the tube cameras.CCD cameras are offered by a number of manufacturers such as DiagnosticInstruments, Inc., Hamamatsu Photonics Deutschland GmbH, Javelin Sys-tems, Kappa MeBtechnik GmbH, PCO Computer Optics GmbH, PolaroidExport Europe, Princeton Instruments, Inc., Proxitronic GmbH, PTI, Inc.,Sony Corp., or Theta Systems GmbH. Colour cameras with either one orthree separate chips for red, green, and blue (RGB signal) which are usefulfor dye discrimination in histology are rarely useful for video microscopy.When selecting a camera make sure that it can be used without automaticgain control (AGC) because gain and offset (pedestal) must be set by theuser manually (see Section 2.3, Table 2, step 5). Only if this is possible, cananalogue contrast enhancement be performed.

The non-uniformity of response across the camera target is called shading.In some low light level cameras this effect can be as high as 20% from one

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Figure8. Cameras and camera control units for video microscopy, (a) B/W vidicon cam-era C 2400-77E 2/3" (Hamamatsu Photonics) and its control unit with gain, offset, andanalogue shading correction capability, (b) B/W intensified (CCD camera C2400-81 Hama-matsu Photonics) and its control unit, (c) Chilled B/W (CCD camera C5985 HamamatsuPhotonics) with control unit (courtesy of Hamamatsu Photonics Corp., USA).

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region to another. A number of commercially available cameras offer'shading correction' which allows the user to introduce various combinationsof linear and parabolic waveforms to compensate for this non-uniformity.Examples of cameras and controls are shown in Figure 8.

1.4.2 Camera systems for video-intensified microscopy (low lightlevel cameras)

Historically, a diverse number of camera technologies have been applied tolow light level imaging. For a variety of reasons most of these technologieshave given way to two general classes of low light detectors: intensified videocameras and cooled solid state cameras. From an application standpoint thesedetectors are differentiated from one another by the fact that the former,being intensified, is capable of viewing dynamic specimens; while the solidstate device, being an integration-type detector, is, at very low light levelsituations, suitable only for very slowly moving or static samples.

i. Intensified video camerasThese devices, as implied by the name, consist of two separate functionalcomponents—an image intensifier and a video camera. The image intensifierserves to detect the image, amplify its intensity, and present the resultingimage to the video camera so that it may be 'readout' in a systematic format.

The most common low light level camera currently utilized in microscopy isthe silicon intensifier target (SIT) camera. This design combines an electro-statically focused image intensifier with a silicon target camera tube within acommon glass envelope. SIT cameras can provide sensitivities up to 100 timesgreater than the silicon target camera alone, which itself is considered asensitive camera. The price to be paid is, however, a considerably lowerspatial resolution.

The SIT camera is also available in a double intensified configurationknown as the ISIT (intensified silicon intensifier target). This camera utilizesan additional intensifier which is fibre-optically coupled to the photocathodeof the SIT. This combination provides sensitivity approximately 20-30 timesgreater than the SIT camera and allows for operation very close to the limitsof human vision. Both the SIT and ISIT normally employ a multialkali photo-cathode which provides spectral sensitivity from 300-850 nm (Figure 9)(11, 17).

An alternative approach to the SIT/ISIT design is to couple optically animage intensifier to a video camera. In contrast to the SIT, most image intensi-fiers employ a phosphor window as the output element, thereby reconvertingthe electron image back to an optical image for viewing by the video camera.By way of lenses or a fibre-optic coupling the image at the phosphor is focusedonto the faceplate of the video camera. A major practical advantage of thisdesign is that it provides flexibility in selecting image intensifiers and videocameras with performance characteristics for specific applications (Figure 8b).

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WAVELENGTH (nm)

Figures, (a) Sensitivity range of vidicons and low light level cameras relative to incidentlight intensity. Very sensitive film is included for comparison as well as illuminance levelsof moonlit and starlit scenes. Photon counting imager type 1 is with phosphor screen out-put of the first stage while type 2 is with semiconductor-based position-sensitive detectoroutput, fc, foot candles or lumens per square foot; lux, lumens per square metre, (b)Spectral responses of the three most common types of photocathodes. B, bialkali type;M, multialkali type (S20); S, S1 type.

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The major differences among image intensifies revolve around the focus-ing mechanisms employed and the method of amplification. The simplest con-figuration is the wafer, or biplanar, type. Its greatest attributes are small sizeand lack of distortion. Higher performance in terms of gain and image qualitycan be obtained by incorporating a focusing mechanism and both electrostaticand electromagnetic focusing systems have been employed. The electrostatic-ally focused type is more compact, lightweight, and less expensive. For extremelyhigh intensification requirements these tubes can be configured so that theoutput phosphor of one is optically coupled to the input photocathode ofanother. Such cascaded intensifiers can realize luminous gains in excess of 106,when constructed of three or four stages.

High gain can also be achieved by providing for electronic amplificationwithin the image tube itself (17). This is made possible by placing a micro-channel plate (MCP) between the photocathode and output phosphor. A singleMCP can provide electron gains of 103 and multiple MCPs may be used forhigher gain requirements. MCP image intensifiers offer similar performanceto multiple stage electrostatic focused systems with smaller size, lower distor-tion, and decreased power requirements (Figure 9a). Combinations of intensi-fied cameras and tube cameras or CCD cameras are available for applicationswhere both highest sensitivity and video rate capacity are required such as forhigh time resolution Ca2+-imaging (Figure 8b). Such combinations reach ex-tremely high sensitivities down to single photon counting range (Figure 9a)sometimes with extremely wide dynamic ranges (up to nine orders of mag-nitude). Manufacturers of SIT cameras, intensified tube cameras, and inten-sified SIT cameras, would include Cohu, Inc., DAGE-MTI, Inc., HamamatsuPhotonics Deutschland GmbH, and Proxitronic GmbH.

ii. Cooled solid state detectorsRecent advances in the development of CCDs have been very promising withregard to their application in low light microscopy. While technically not anintensified camera, the CCD camera has the ability to obtain images at lowlight levels. Being inherently high quantum efficiency devices with respect tothe ability to convert photons into electrons, high sensitivity is achieved pri-marily by cooling and slow readout. Cooling the CCD (Peltier cooling toaround 0°C or external cooling to - 125°C) dramatically reduces the dark cur-rent as a noise component. The slow readout further reduces the noise associ-ated with high bandwidth electronics. Low light images are integrated directlyon the chip in much the same way as an extended photographic exposure. Highquality slow-scan CCDs offer excellent geometry, photometric accuracy, andlarge dynamic range and are, therefore, the cameras of choice in microscopyfor the quantitative imaging of static low light samples. These cooled CCDdetectors require their individual special imaging board to guarantee precisereadout of the CCD chip which is achieved only at relatively slow rates. Whileformerly one image required up to several seconds, 5-10 Hz cameras are

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presently available and video rate is expected to be possible in the near future.Hamamatsu has recently developed cooled CCD cameras (e.g. HamamatsuC5985) achieving readout rates of seven images per second (Figure 8c). Thiscamera type could, therefore, provide a good alternative to SIT camerascurrently used in dynamic VIM. Manufacturers of suitable slow-scan CCDcameras are Diagnostic Instruments, Inc., Hamamatsu Photonics DeutschlandGmbH, Javelin Systems, Kappa Messtechnik GmbH, Optronics Engineering,PCO Computer Optics GmbH, Photometries Ltd., Photonic Science Ltd.,Princeton Instruments, Inc., PTI, Inc., Theta Systems GmbH, and others. Forfurther information about cooled CCD cameras see refs 11, 16, and 18.

Because cooled CCD cameras are controlled via a specific computer inter-face (image acquisition board), one should first decide on the computer plat-form (Macintosh-, PC-, or Unix-based) to be used with the camera system.Most manufacturers will offer solutions for both Macintosh and PC com-puters. By deciding on a particular computer platform one should, however,only consider computers with a fast processor and sufficient RAM (minimumof 64 MB) and hard drive memory (minimum of 2 GB).

Image acquisition requires dedicated software packages compatible with avariety of slow-scan cameras and their specific interface boards, such as Meta-Morph (Universal Imaging Corp.), IPLab Spectrum (Scanalytics, Inc.), NIH-Image (public domain from NIH, Bethesda), AxioVison (Carl Zeiss GmbH),or the packages obtainable from Data Translation, Inc. or Vaytec, Inc. Someof these software packages also control video recorders in a microscope work-station and can be used to improve image quality. PhotoShop (Adobe Systems,Inc.) and Paint Shop Pro (Jasc Software, Inc.) are general software programsto improve image quality, but are not sufficient to control sophisticatedcameras such as cooled slow-scan CCDs. A detailed comparison betweencooled CCD and SIT cameras is provided in ref. 11.

Hi. Practical considerations in choosing a low light level camerasystem(a) Sensitivity. Clearly the most important consideration in selecting a

camera for low light imaging is sensitivity. A wide variety of cameras isavailable with sensitivities ranging from applications in DIC and phase-contrast microscopy down to the single photon level. Sensitivity is typi-cally measured by illuminating the camera target with a known quantityof tungsten light and measuring the resulting output current of the camera.The data is expressed in amperes/lumens and is plotted in a log-log fashionas the 'light transfer characteristic'. This provides a useful way to comparevarious systems but this definition can be misleading when applied to lowlight cameras. It does not take into account the noise component of theoutput signal and, being based on tungsten light, is heavily biased towardred-sensitive photocathodes. Figure 9a illustrates the typical sensitivityrange of a number of low light level cameras.

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(b) Spectral response. The spectral response of intensified cameras is deter-mined by the window material and the type of photocathodes. The mostcommon photocathode is the multialkali type (S20) which has a peaksensitivity at 420 nm and provides usable sensitivity to approximately800 nm. The spectral responses of three common photocathodes, multi-alkali, bialkali, and SI, are shown in Figure 9b. It should be noted that theextended red-sensitivity of the S20 and S1 types is accompanied by highernoise levels (thermal noise). While not a problem in most situations, atvery low light levels one is advised to select the bialkali type unless thisextended sensitivity is required.

(c) Resolution. As noted, high resolution and high sensitivity tend to bemutually exclusive characteristics. This relationship is due to noise. Noisein low light level systems can generally be classified as signal-independentor signal-dependent. Signal-independent noise is essentially present in afixed amount, in both the absence or presence of light. It arises fromthermal noise of the intensifier, the camera target, the video-amplifier,etc. This is the predominating type of noise at relatively high light levels.As light levels decrease signal-dependent noise becomes the dominantcomponent. At low photocathode illumination levels, resolution isprimarily limited by the finite number of photoelectrons released at thephotocathode—the so-called photoelectron, or quantum, noise. It is thislatter noise which accounts for the decrease in resolution as light levelsdecline. Resolution, therefore, should be evaluated as a function of lightintensity and preferably at a specific wavelength when comparing forpossible photocathode differences.

(d) Lag. Lag, or dynamic response, describes the camera's ability to respondtemporally to changes in light intensity. If the system is to be used for theimaging of rapidly changing specimens, a camera with low lag character-istics should be selected. Lag in an intensified camera is due primarily tothe readout video camera, therefore utilizing a low lag camera such as asaticon or solid state will greatly improve this characteristic. One shouldbe aware that certain camera types retain a considerable part of the in-formation (up to 30%) from one frame to the next. For more details seeref. 17.

Microscope/camera combinations suitable for both VIM and VECmicroscopy are depicted in Figure 10.

1.4.3 Analogue image processorsAnalogue image enhancement is the step in video microscopy whereby mostof the possible image improvement is gained. Analogue functions (Figure 1)are either incorporated into the camera control units (Figure 8) or into theframe grabber boards. Essential for video microscopy is the capability to man-ually set gain and offset within a wide range. Cameras with automatic gain

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Figure 10. (a) Video microscopy set-up based on a Zeiss Axiophot. The monitor for theprocessed image is seen behind the Hamamatsu control unit C2400 for SIT camera (front)or Newvicon camera (back). The control unit can be used for either one of the two cam-eras unless simultaneous use of both cameras is needed (courtesy of Hamamatsu Pho-tonics Corp. USA), (b) Multi-purpose video microscopy workstation using a NikonDiaphot 300. (1) Newvicon Camera (Hamamatsu C2400-08); (2) fan heating system(Nikon) for 37°C box around the stage; (3) mercury HBO100 lamp house; (4) XenonXBO100 lamp house with remotely controlled shutter (Uniblitz); (5) low light camera SIT(Hamamatsu C2400-07); (6) slow-scan camera (Photometries SenSys); (7) control unit forSIT camera with ARGUS 10 and control unit for Newvicon camera with ARGUS 20(Hamamatsu Photonics); (8) video monitor, Uniblitz control unit and time lapse videorecorder (Panasonic AG6730 SVHS); (9) colour printer (Tektronix Phaser 350); computer(Macintosh PowerMac). The set-up can be used simultaneously for VIM and VECmicroscopy because the high resolution (5) and low light (6) cameras are connected to aNikon multi-image module containing a proper dichroic beam splitter.

control (AGC) only or gain and offset adjustable only in a narrow range or bysmall set screws on the back of the camera are, therefore, unsuitable. Somecontrollers provide additional analogue processing features for image pre-processing such as shading correction, intensity line scan, and others. As a rule,cameras should fulfil the requirements mentioned in Section 1.4.1 especiallywhen offered for microscopy applications. Suitable equipment is provided byColorado Video, Inc., DAGE-MTI, Inc., For-A Company Ltd., HamamatsuPhotonics Deutschland GmbH, or Optronics Engineering.

1.4.4 Compact digital image processorsDigital image processors for video microscopy may come as (i) compactstand-alone systems, which also may be (ii) externally driven together withother microscopy devices (stage motor, filter wheels, etc.) by a PC, or (iii)fully PC-based systems (see Section 1.4.5).

Compact digital image processors are the minimal requirement available atreasonable cost for those who need digital image processing in real time inorder to obtain the desired image quality, but do not intend to do further digitalimage analysis (e.g. morphometry) of single frames with the same system. Beaware, however, that some of these systems do not include the analogue en-hancement feature, which is indispensable, if cameras without their ownanalogue enhancement capability are to be used. The processors most suitablefor our purpose should include digital enhancement, real time subtraction withsimultaneous frame averaging functions, plus additional potentially usefulfeatures. Within this group ARGUS 10 and 20 (Hamamatsu Photonics) arethe most widely used device (Figure 11).

A few dedicated systems are available which allow all these essential realtime functions and provide the user in addition with a fair number of measure-ment functions and some flexibility to manipulate and analyse a set of storedimages. To this end these stand-alone systems may be connected to an externalPC and thus allow to access software libraries for specific applications. These

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Figure 11. Compact image processor for analogue contrast enhancement and digital pro-cessing, (a) Example of the mouse-driven on-screen menu of the Hamamatsu ARGUS 20(b) that permits real time image improvement by a great variety of both analogue anddigital functions together with additional measuring functions (bottom).

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systems can provide software for real time image processing (e.g. for ratioimaging) and for controlling additional hardware such as video recorders, filterwheels, or special cameras. In combination with remotely controlled highspeed shutters (e.g. Uniblitz) and video recorders important features arise:time lapse recording (each n-th frame), or alternate recording from twocameras, such as continuous DIC recording with one and fluorescence with asecond camera. Generally, previously recorded sequences played back from arecorder can equally be processed with these systems. Examples in thiscategory are the ARGUS 20 as a stand-alone system or the PC-drivenARGUS 50 series of Hamamatsu Photonics and the systems from OptronicsEngeneering or Datacube, Inc.

1.4.5 PC-based digital image processorsThe improving speed and storage capacity of PCs now in use allows them tobe used with appropriate analogue-to-digital converters (A/DC), which areoften integrated in sophisticated frame grabber boards, to perform the requiredsteps for video microscopy. A whole industry offers image processing boardsand software packages that perform both image acquisition and processingnot only with single frames but also at video rate. Examples of companies pro-ducing frame grabber cards are Datacube, Inc., Data Translation, Inc., FastElectronic GmbH, HaSoTec GmbH, Imaging Technology, Inc., Matrox Elec-tronic Systems Ltd., Scion Corp., or Silicon Graphics, Inc. For high perform-ance tasks such as pixel point processing in single frames (digital spatial filters,Fourier transforms) or two- and more-frame processing (ratio imaging, 3Ddisplay) dedicated on-board processors are widely used, although the rapidprogress in computer performance makes fully software-based systems ofastounding capabilities already possible.

A dedicated system would have to include all the above mentioned process-ing functions and the capacity of controlling ancillary devices such as a varietyof commonly used cameras including slow-scan CCD cameras, focus motor,shutters, stage drive, and motorized filter wheels or filter sliders. Very import-ant is the capability to synchronize one or two recorders with cameras andshutters in order to allow parallel recording and to protect the fluorescentspecimens from photobleaching by recording only a few frames after every n-thminute in the fluorescence channel. Such regimes can be very helpful becausethey permit long-term experiments without extensive photobleaching. Thesystem should also offer a number of software routines needed for biologicalvideo microscopy, such as for ratio imaging, organelle tracking, or multiplefluorescence. The display functions should include playback of stored videosequences in real time and in time lapse mode, i.e. selecting each n-th frameand assembling it into video sequences (video-clip). For post-processing theavailability of point, kernel, and frame operations for further image improve-ment and extraction of quantitative data (data analysis, morphometry) aredesirable.

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Several companies offer fast or video rate image processing systems work-ing on PC-, VME-, or UNIX-based platforms that combine almost all of theabove mentioned features (e.g. Bitplane AG, Improvision, Inc., InovisionCorp., Media Cybernetics, NIH-RSB, Scanalytics, Inc., Silicon Graphics, Inc.,Stemmer Imaging GmbH, Universal Imaging, Inc.). Some companies alsooffer software packages capable in addition of very useful device control (videorecorder, light shutter, etc.) and microscope automation features (Carl ZeissGmbH, Inovision Corp., Scanalytics, Inc., Universal Imaging Corp., VayTec,Inc.). A high-end IBM compatible PC system is that developed by UniversalImaging Corp. using their software package MetaMorph (19). Alternatively aMacintosh-based system as that depicted in Figure 10b could be assembledfrom available components.

In our case this consists of a Nikon Diaphot 300 microscope with DICoptics and epifluorescence equipped with four different cameras attached tofront, side, and two upper ports. We are currently using a 35 mm Nikon F-601camera (front port), a SIT camera (Hamamatsu C2400-08) with controllerconnected to an ARGUS 10 image processor (side port), a NIKON multi-image module (upper port) with two cameras, a Newvicon camera (Hama-matsu C2400-07) with controller connected to an ARGUS 20 imageprocessor, and a SenSys cooled digital CCD camera (Photometries Ltd.) withLG-3 computer interface card (Scion Corp.). The multi-image module allowsby using an appropriate dichroic mirror to use epifluorescence and DICsimultaneously (20).

In our set-up two ARGUS image processors (ARGUS 10 for SIT cameraand ARGUS 20 for Newvicon camera) are used for digital enhancement, sub-traction, and frame averaging. Image acquisition is performed from theARGUS processors either by a Panasonic S-VHS time lapse video recorderor directly by a Power Macintosh 9600 (350 MHz/128 Mbyte RAM/3 GB HD)equipped with the LG-3 frame grabber (Scion Corp.), which captures grey-scale images and provides four input channels that can be used for imageacquisition from different cameras. The S-VHS time lapse video recorder(Panasonic, model AG-6730) is controlled via a RS-232C interface adapterfrom the computer serial port. This combination is able to count and accessindividual frames' positions on the tape.

The image acquisition software used is IPLab Spectrum (Scanalytics, Inc.)which supports, in our case, (i) the LG-3 frame grabber, (ii) the slow-scancamera (SenSys, Photometries Ltd) interface hardware, (iii) the video taperecorder control, and (iv) the shutter control (Uniblitz).

Using the IPLab Spectrum software in combination with the appropriatehardware it is possible to control several video cameras and video recordersand capture images onto the computer. Using a microscope set up with aNewvicon camera and a SIT or cooled CCD camera one can record DICimages and fluorescence images simultaneously. If time lapse series of fluor-escence images have to be recorded it is highly recommended that a Uniblitz

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shutter be included to protect fluorescent specimens from photobleachingduring periods of no recording. All of these features can be controlled withthe IPLab Spectrum software in combination with the LG-3 frame grabbercard (Scion Corp.).

Examples for studies that can be performed with this set-up include:

(a) Tracking of organelle movement along fluorescent actin filaments in vitro.Fluorescent actin filaments labelled and stabilized with rhodamine-phalloidin are visible in the fluorescence channel and moving organellesin the transmitted light DIC channel (21).

(b) Tracking of organelle movement along fluorescent microtubules in livingcells. Microtubules labelled by microinjection of rhodamine tubulin intothe cells are visible in the fluorescence channel and moving organelles inthe transmitted light DIC channel (see also refs 7 and 20).

1.4.6 Single function processorsWhile the above mentioned devices are more or less multifunctional, some-times only one or two functions may be required. In this case it may be cheaperto obtain unifunctional, hard-wired instruments for analogue enhancement,distance measurement, perimeter measurement, time/date generation, orintensity measurement along a scan line etc. Such devices can be obtainedfrom, for example Colorado Video, Inc., For-A Company Ltd., HamamatsuPhotonics, or Optronics Engeneering.

1.5 Considerations on the microscopeFor all types of video microscopy, a high performance research microscopeshould form the basis of the optical imaging system. Due to the unusually highmagnifications used for video microscopy special attention has to be paid tobright illumination, optimized light throughput, and high mechanical micro-scope stability.

A heavy stand for the microscope is recommended in order to reduce vibra-tions and internal movements resulting from temperature changes. This isespecially important when heavy cameras are used like those designed forVIM and when using highest magnifications. It should be noted that a 1 umdisplacement in the specimen is registered on the video screen at typical magni-fications for VEC microscopy as 1 cm. In some cases vibration isolation tablesmay therefore be required.

1.5.1 IlluminationVideo cameras must work near the saturation end of their dynamic range inorder to enable one to utilize their contrast enhancement advantages. Whenplanning high magnification VEC microscopy xenon or mercury arc lamps arerequired, in most cases, to provide enough light, so that the cameras worknear the saturation end. Furthermore, it may be essential that all unnecessary

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components such as filters and diffusers are removed from the light path andthe lamp is optimally adjusted. Only at lower and intermediate magnificationsor in some microscopes that are optimized for very high light transmission 100W halogen or tungsten lamps may be sufficient even for VEC-DIC. We haveseen the best results with the HBO 50W DC mercury lamp, which has a shortintense arc, in combination with the Axiomat and the Axioskop (Carl ZeissGmbH), the inverted microscopes IM and ICM (Carl Zeiss GmbH) and thePolyvars (Reichert/Cambridge Instruments). The 100 W or 200 W mercuryarc lamps also worked well in most cases, although sometimes the former mayhave a too small bright centre, thus producing a central hot spot (Figure 6),while the latter may not be bright enough because the light is too much spread.Great care should be taken to set the illumination so that it is uniform andfilling the NA of the condenser. If this is not possible with the arc lamp used, afibre-optical scrambler may be inserted (see ref. 2, p. 127).

Due to the fact that after contrast enhancement minute changes in lampintensity may result in transitions from well modulated to bright white orblack images, stabilized DC power supplies may be unavoidable. With arclamps it is advisable to use a narrow-band green interference filter (e.g. theHg line 546 ± 10 nm for mercury lamps) for optimal DIG results and to pro-tect cells from blue light. In this case it is also necessary to protect the inter-ference filter, the polarizers, and the cells from heat and UV light with at leastone piece of each of the following filters: a UV filter, a heat-reflecting filter,and a heat-absorbing filter.

1.5.2 Optimized light gathering and throughputAll VIM and most of the high magnification VEC microscopy applications arecharacterized by the limited amount of light available. Therefore, by far thetwo most important considerations involve optimizing the microscope for lightgathering ability and the efficient transmission of light through the optical path.

The light gathering ability of the microscope is a direct function of thenumerical aperture (NA) of the objective lens. While factors such as an object-ive's working distance often necessitate using a lens with lower than maximalaperture for a specific magnification, whenever possible the highest availablenumerical aperture should be utilized. The importance of this point is clearlyappreciated when one considers the relationship of magnification, numericalaperture, and light intensity. In a system where the illuminating aperture isequivalent to the objective's acceptance aperture (such as in epifluorescence),intensity (/) is proportional to the fourth power of the numerical aperture.

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Further, light intensity is inversely proportional to the square of the magni-fication:

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Therefore:

In comparing two X 40 objectives with apertures of 0.9 and 0.5, onesees that the higher numerical aperture objective captures over ten timesmore light than its lower numerical aperture counterpart. The demand ofhigh NA objectives necessitates the use of oil or water immersion objectivesand condensers whenever possible. It should be clear to the reader that theultimate performance of VEC-DIC (e.g. visualization of microtubules) willrequire a X 63 or X 100 objective with NA 1.3 or 1.4 and a oil condenserwith NA 1.4.

As a general rule, in lenses of similar design, the numerical aperture tendsto increase with magnification. The above relationship clearly dictates thatwhen using additional magnifying optics, such as optovars, relay optics, orprojection eyepieces, one should maximize the numerical aperture and mag-nification of the objectives, and minimize the magnification of the intermedi-ate optics, since these optics contribute nothing to the light gathering abilityof the system. Therefore, if faced with the choice of using a X 40 objective incombination with a X 10 camera relay lens versus a X 25 objective and X 16relay lens, the former combination will prove far more efficient despite thefact that both systems deliver a final magnification of X 400.

Regarding the second major consideration, we have to remember that theefficiency of light transmission within the optical system is primarily a func-tion of the transmission properties of the objective lens and the number ofoptical elements in the system. Interestingly, there are often significant differ-ences in the transmission characteristics of objectives of different designs (e.g.fluorite versus apochromatic) and from different manufacturers, even whenthe numerical aperture and magnification are identical. If possible, it is recom-mended that a number of lenses be evaluated with regard to the specific wave-length(s) that will be utilized.

Of equal, if not more, importance, is the issue of intermediate optical ele-ments. Uncoated lens surfaces typically reflect 4—5% of the light incidentupon them. And while it is true that research quality microscopes generallyemploy anti-reflection coatings, these cannot completely eliminate losses dueto reflection and the cumulative loss associated with the complex light pathscan be substantial. Therefore, all unnecessary optical elements should beremoved from the light path. If this is not possible, the use of a microscopewith a simple light path should be considered.

When planning fluorescence work, make sure the optics transmit the shortwavelength light which is often required for the excitation of dyes, for exam-ple 340 nm for Fura-2 used for video measurements of Ca2+ concentrations.The requirement of the highest quality optics is sometimes even less stringent,since in most cases monochromatic light is recommended anyway, and since

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often only the central area of the field (~ 1/3 to 1/2) is picked up by the videocamera. Wide-field optics are usually of no particular advantage.

1.5.3 Special considerations for AVEC microscopyFor AVEC microscopy (see Section 2.1) two special considerations arcimportant:

(a) De Senarmont compensator. In order to set the recommended bias retar-dation of 1/9 of a wavelength (546 nm)a de Senarmont compensator set-upis required (see Section 2.3.4). Most biological DIC and POL microscopeshave only the Brace-Kohler type or other compensators or analysers thatallow only the introduction of bias retardations of 1/100 or 1/50 of a wave-length but not 1/9. Nikon is the only manufacturer to offer a dc Senar-mont compensator for AVEC microscopy routinely with their Eclipsemicroscopes. It consists of an additional quarter wave plate (y/4 plate)which converts the polarizer or the analyser into a compensator (22). The\/4 plate should match the wavelength used, preferably the green Hg line(546 nm). Other microscope manufactures will, however, have the properparts in their mineralogical programmes (Figure 12).

Figure 12. Parts for AVEC-DIC microscopy with de Senarmont compensation as built intoa Zeiss Axiophot microscope. (1) Objective Wollaston prism; (2) 1/4 wave plate; (3) X 1.6magnification optovar instead of slider for the fluorescence filter blocks; (4) rotatable cal-ibrated polarizer used as analyser; (5) additional magnification changer x 2.5. With thissetting an empty camera attachment tube can be used. Alternatively, a fixed x 4 cameraadapter tube can be used instead of the two optovars to obtain the additional magnifica-tion. The polarizer and Wollaston prism in the condenser are not visible,

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(b) Short shear Wollaston prism. In DIC microscopy visual contrast generationdepends on the shear introduced by the pair of Wollaston prisms. If, how-ever, the shear is too large, i.e. larger than the resolution, not only the brightand dark shadow-cast margins that make objects visible at high contrast, butalso annoying slightly shifted double images will appear. For work with theeye best contrast is achieved at a shear just below its limit of resolution (i.e.200 or 220 nm). For video microscopy that is higher in resolution, so that200-220 nm are truly resolvable, shorter shear prisms are required to makeuse of the gain in resolution. Therefore, shorter shear sets of Wollastonprisms are desirable to fully utilize the advantages of AVEC microscopy.Nikon is the first manufacturer to acknowledge the importance of special'short shear' prisms for VEC microscopy as it offers two sets of prisms withtheir Eclipse microscopes: 'short shear' prisms with low visual contrast buthigher resolution with video enhancement and 'medium shear' prisms forhigh contrast observation by eye but lower resolution with VEC techniques.

1.5.4 Additional magnificationFor video microscopy we need considerable additional optical magnification.This is due to the fact that because of the relative small number of video linesthe video image has a spatial resolution which is much lower compared to thatof a photomicrograph or that of the microscope optics. In order to fully utilizethe resolution of the microscope we have to make sure that it is the limitingcomponent rather than the video system. This means that we have to magnifythe specimen onto the target of the video camera far beyond the magnifica-tion which is normally considered useful in conventional light microscopy. Fora detailed discussion of object resolution matching the CCD pixel size seeref. 23. Whenever possible, this should be reached by the use of higher powerobjectives, but when subresolution objects are to be visualized with the X 63and X 100 oil immersion objectives, additional magnification of X 4 to X 6.3is required. This can be achieved either by a zoom system as in the ZeissAxiomat and the Zeiss Axio series, or by a X 2 or more additional magnifica-tion changer (optovar-type) and/or a high photo eyepiece (X 16 or X 25) plusprojective lens (50-63 mm) as in most other microscopes, e.g. IM, ICM, andAxio series (Carl Zeiss GmbH), Orthoplan and Aristoplan (Leitz), and Poly-var (Reichert/Cambridge Instruments). Alternatively a combination of twoX 2 or one X 4 extension tubes may be used (Zeiss, Leica, Nikon). As a ruleof thumb, when objects at or below the limit of resolution are to be observedon a medium-sized monitor, the desirable maximal useful magnificationshould be such that the field of view portrayed on the monitor is 15-30 u,mwide, or the final magnification is around X 8000 to X 15000.

1.5.5 How to interface microscope and cameraOne should ensure that 100% of the light can be directed to the camera. If themicroscope has a fixed 'split' (80/20% or 50/50%) between the binocular and

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camera port, it may be advisable to have a 100/0% reflecting mirror installedon the slider in place of the existing one.

The system projecting the images onto the camera target classically con-sisted of the ocular and the camera objective lens in close contact. Both con-sist of several lens elements and often some surfaces are close enough to theimage plane to introduce very disturbing mottle from dust, grease, and non-perfect coatings. Oculars or projecting oculars are, however, indispensable inall cases where part of the correction of optical aberrations is taken care of inthe objective lens and part in the ocular.

Several manufacturers have switched to totally internally corrected object-ive lenses, so that microscopists have much more freedom to use alternativearrangements. In such optical systems the tube lens directly projects an inter-mediate image onto the TV target through an empty connecting tube. Thisyields very good images with little mottle. For highest magnifications, thisarrangement requires that the additional magnification of at least X 4 isreached by a Galilean-type telescope in the parallel, infinity corrected, light path,or by an additional magnification changer ('optovar'), or by a combination ofboth.

Another connection, which creates less mottle but requires considerablebench space, is to mount the camera to a lateral exit with a high power eyepieceand to project the intermediate image directly onto the camera target. Thedesired magnification is adjusted by sliding the camera to and from the micro-scope at a 10-30 cm distance.

For biological microscopy it is very important to note that monochromaticDIC and fluorescence images of different wavelengths can be sent to two dif-ferent cameras simultaneously by using a proper dichroic beam splitter (20).Using the multi-image module (Nikon) (Figure 10b) one can increase thenumber of detectors mounted simultaneously. By using an appropriatedichroic mirror it is possible to direct all the emitted light from the epifluor-escence to a SIT or slow-scan camera (wavelength 1) while all the light fromtransmitted light DIC goes for simultaneous imaging of fine detail to a New-vicon camera (wavelength 2). Intermediate magnification lenses (X 1.25, X1.5, X 2.0) inside the multi-image module and the C-mount zooming adapter(Figure 10b, upper left) allow the fluorescence and transmitted light images tobe adjusted to the same magnification independent of camera type and targetsize.

2. High resolution: video-enhanced contrastmicroscopy

2.1 Different types of video-enhanced contrast microscopyVideo contrast enhancement of microscopic images using bright-field, dark-field, anaxial illumination, or fluorescence techniques is very straightforward

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INTENSITY

Figure 13. Dependence of intensity (image brightness) and contrast (see Section 1.3.3) ofDIC images of phase objects on phase retardation. Phase retardation is introduced andvaried by laterally displacing a Wollaston prism or by setting the de Senarmont compen-sator away from extinction. This converts positive and negative phase gradients of speci-mens to contrast, thus producing highlights (/H) and shadows (/s) relative to a neutralgrey background (/B). A phase shift of n/2 radians corresponds to \/4, i.e. 1/4 of a wave-length. It can be seen that theoretically the image contrast is highest at the position n/2(vertical arrow). Due to straylight the image background (/B) is of considerable intensity atthis setting and has to be compensated for by the addition of a negative voltage (offset).For reasons discussed in the text, smaller retardations (1/9 of wavelength) are recom-mended. (Reproduced with permission from ref. 4.)

and can generally be described by the term 'VEC 'microscopy'. Allen (3, 4)and Inoue (1) simultaneously described procedures of video contrast en-hancement for polarized light techniques which differed considerably in theirapproach but yielded very similar results. There is a need to distinguishclearly between the two strategies in order to avoid confusion.

Allen named his techniques 'Allen video-enhanced contrast' differentialinterference contrast and polarization (AVEC-DIC and AVEC-POL, respect-ively) microscopy. These techniques involve setting the analyser and polarizerfar away from extinction in order to gain a high specimen signal IS (see Sec-tion 1.3.3). Allen suggested the use of a de Senarmont compensator set-up (3, 4,22) which is comprised of a 1/4 wave plate (specific for the wavelength used)in front of a rotatable analyser (Figure 12). He recommended a bias retarda-tion of 1/4 to 1/9 of a wavelength away from extinction with 1/9 as the bestcompromise between high signal and minimal diffraction anomaly of the Airypattern. The enormous amount of straylight (IB) introduced at this setting(Figure 13} is removed by adding the appropriate negative offset voltage.

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The technique recommended by Inoue, which in this chapter is called IVECmicroscopy for the purpose of distinction, aims to reduce straylight and dif-fraction anomaly arising mainly from curved lens surfaces (Table -1) by employ-ing extremely strain-free objectives and the rectifying lenses developed byInoue (24). The latter were commercially available some time ago and onlyfor a few microscopes, such as some lines of Nikon. Inoue's microscope (2, 24)is optimized in such a way and works at a polarizer setting very close to extinc-tion, which cannot be used for VEC microscopy with most other instrumentsbecause insufficient light for near-saturation is passed to the camera. In IVECmicroscopy, straylight is not admitted since the polarizers stay close to extinct-ion and the special rectified optics further reduce the straylight. Consequently,filters to reduce the brightness are not required. On the contrary, a bright arclamp, ideally with a fibre-optic illuminator, is necessary to saturate thecamera.

The AVEC technique electronically improves optical images of low con-trast and high straylight content due to 'non-optimal' optical arrangement,while in IVEC microscopy no compromise is made regarding the optics andconsequently less demanding electronic steps are required to rescue the image.The AVEC technique is, however, the only one which can be used with anygood research microscope equipped with commercial film polarizers. Theproper compensator setting can be experimentally evaluated between 1/100and 1/4 of a wavelength within the ability of ones illuminating system tonearly saturate the camera. Best resolution is achieved on theoretical groundsat 1/9 of a wavelength (25) and best visualization (highest contrast) has beenreported for 1/15 of a wavelength in some microscopes (26). However, accord-ing to Allen the dimensions of an object imaged at the latter setting may differconsiderably, if it is orientated in different directions due to the diffractionanomaly of the Airy pattern (3, 4).

2.2 Sample preparationSamples used in conventional light microscopy can also be used for videomicroscopy. Live cells from tissue cultures should preferentially be grown ona coverglass. The specimen's region of interest should be close to the cover-glass surface, where the best image is obtained. If highest magnifications areintended, it may be found that the optics can be adjusted for Kohler illumina-tion only at this surface and about 20 um below (upright microscope), sincehigh magnification objectives are usually designed for optimum imaging ofobjects at a distance of 170 nm from the front element (No. 1 glasses, approxi-mately 170 um thick). To identify objectives for which this distance and thepresence of a 170 um thick coverglass is critical, look for the '0.17' engravedor refer to the manufacturer's data sheet. If thick cells or other extended spec-imens must be observed with such objectives, thinner glass slides (0.9 insteadof 1 mm) may have to be used instead of the regular ones to allow the setting

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Figure 14. Microscope flow chamber suitable for highest magnification VEC microscopywith upright and inverted microscopes and for superfusion of live cells or suspensionspecimens. (1) Metal frame of the size of regular microscope slides made of brass or alu-minium about 2 mm thick and absolutely flat with an insert to hold a 24 x 50 mm cover-glass (2), The coverglass can be secured to the metal frame by placing small drops ofVALAP (3) at the corners of the insert. (4) Small coverglasses or thin adhesive tape can beused as spacers (e.g. double sticky Scotch TapeR). (5) Coverglass (e.g. No. 1; 24 x 24mm). (6) Filter paper wick to induce medium flow (arrow).

up of Kohler illumination with oil immersion of both condenser and objectivelens. If necessary, No. 0 coverglasses (80-120 um thick) can be used (bothglass slides and coverglasses are available from O. Kindler GmbH or ClayAdams Co.), but it should be noted that under the latter condition imagequality may be impaired. The use of a slide preparation made of two cover-glasses mounted in a frame as depicted in Figure 14 would serve the samepurpose.

For imaging deep within an aqueous sample, water immersion objectivesmust be used. Special high NA objectives for working with aqueous speci-mens have recently become available from manufacturers for confocal micro-scopes. These lenses combine high numerical aperture (e.g. x 60, NA 1.2) withcorrection for 'extreme' working distances of up to 220 um below the cover-glass (27). Note, however, that, if regular oil immersion objectives are focusedthrough water (the specimen) rather than through glass and oil only, sphericalaberration will be introduced. The use of monochromatic illumination or ofimmersion oil of different refractive indices (R. P. Cargille Laboratories) isrecommended to overcome this problem.

Aqueous samples have to be prevented from drying out by completely scal-ing the coverglass to the slide. Nail polish may be used or, if live or solvent-sensitive specimens, such as microtubules, extruded cytoplasm, or culturedcells are observed, VALAP is to be used. This consists of equal parts byweight of vaseline, lanolin, and paraffin (melting point 51-53 °C); it liquefiesat around 65°C and is applied around the coverglass with a cotton tip applica-tor. If the specimen is in suspension (e.g. organelles), no more than 5-10 ulaliquots should be used with 22 X 22 mm coverglasses in order to producevery thin specimens (~ 10 um) for best image quality.

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If working with an inverted microscope, the slide has to be inverted withthe coverglass underneath. With most microscope stages this will interferewith the VALAP sealant and flat positioning of the slide will not be possible.It is recommended instead to use a metal frame of the size of a regular slideand 0.8-1 mm thick. A larger coverglass is attached with VALAP so that itcovers the opening. After the sample has been applied, the top coverglass ofregular size and thickness is added with or without spacers made of coverglassor adhesive tape and sealed (Figure 14).

If thick specimens such as tissue slices, vibratome sections, or nerve bundlesare to be observed at high magnifications that require immersion, only con-trasting techniques that allow optical sectioning such as DIC, Hoffman modu-lation contrast, or anaxial illumination (28) (including Varel contrast) arerecommended. Only the first 10-20 (um behind the coverglass will yield perfectimages. However, even if expensive long working distance water immersionobjectives are not available, part of the loss of quality can be compensated forby video enhancement with good results down to a depth of 100 (um or more,with respect to both visibility and contrast, sometimes also to resolution (28).The opacity of live tissue such as brain slices is greatly reduced when light of700-800 nm is used because they are transparent for near-infrared or infraredlight (29).

2.3 Procedure for image generationBecause VEC microscopy requires some steps which are different from con-ventional microscopy, image generation is discussed here in some detail(Table 2). Steps 1-5 of the procedure yield the image if only analogueenhancement is required. The continuation leads to highest resolution andvisualization of submicroscopic objects. The procedure chosen is basicallythat used for AVEC-DIC and AVEC-POL, but if these are not required step4 can be disregarded.

2.3.1 Focusing the specimen: step 1Find the specimen preferably by looking through the oculars or, alternatively,by looking at reduced magnification at the monitor. If the entire specimenconsists of subresolution size material (density gradient fractions, microtubulesuspensions, unstained EM sections) (see Figure 3) it will be difficult to findthe specimen plane. Use a relatively dark setting of the condenser diaphragmand/or polarizers or prisms and look for brightly shining contaminatingparticles. If there are none, routinely apply a fingerprint to one corner of thespecimen side of the coverglass and use this for focusing.

2.3.2 Adjusting Kohler illumination: step 2After finding a coarse setting for the illumination, the desired plane for thespecimen is selected exactly. Then the condenser is finely adjusted, but now inrelation to the image on the monitor (make sure the light is reduced to avoid

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Table 2. Steps in AVEC-DIC microscopy

Step Equipment Manipulation

1a Microscope Focus specimen2 Microscope Adjust microscope correctly for

Kohler illumination3 Microscope Open condenser diaphragm fully

4 Microscope Set compensator up to 20° fromextinctionb

5 Camera Analogue-enhance by manuallyadjusting gain and offset

6 Microscope Defocus or move specimen laterallyout of field of view

7 Processor Average and store mottle image, thensubtract mottle image from incomingvideo images

8 Microscope Return specimen to focal plane

9 Processor Contrast-enhance digitally(histogram stretching)

10 Processor Use rolling or jumping averaging ordigital filtering

11 Processor Spatial filtering using various masks

Result

Image appearsImage improves

Optical image becomes toobrightOptical image worsens

High-contrast video imagewith often disturbing mottlepattern appearsObject disappears, mottleremainsAbsolutely homogeneous,light grey ('empty') imageappearsClear image appears; ifcontrast is weak go to step 9Contrast becomes optimal;if pixel noise is high go tostep 10Clear, low noise, and highcontrast image appearsSharpened image orhighlighting special aspectsof image contents

aBefore step 1, one should set the 'brightness' and 'contrast' controls of the monitor showing the pro-cessed image to their intermediate positions because the degree of enhancement will not be adequatein the recorded sequence if the monitor had been adjusted to an extreme setting. To use VECmicroscopy to its full extent make sure that the microscope objective and condenser front lensesare absolutely clean (check at least once daily) and that the lamp is always optimally adjusted andcentred.* Particles need to appear in DIC microscopy images as if illuminated from above, i.e. with their brightpart up, while vacuoles have the opposite shadows. If this is not the case, the camera has to be rotated180° or the compensator or Wollaston prism has to be set to the opposite side with respect to theextinction position. Modified from ref. 30.

damage of the camera!). The field diaphragm must be centred on the monitorand opened until it becomes just invisible. If the field diaphragm is opened toomuch, most microscope-camera adapter tubes or high power projectives andoculars will create a very annoying central hot spot (Figure 6). If this problempersists after the adjustment for proper illumination, closing the projectivediaphragm, or inserting a self-made diaphragm to cut the peripheral light atthe microscope exit usually helps. Note that at higher magnifications andnumerical apertures, Kohler illumination has to be readjusted once the focusis changed more than a few micrometres.

As we will apply extreme contrast enhancement later, we have to start out

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with as even an illumination setting as possible. Proper centring of the lampand setting of the collector lens are, therefore, important. Also, as much lightas possible needs to be collected at high magnifications. Some workers haveused critical illumination that is focusing the light source onto the specimenplane instead of Kohler illumination (26). This is counter to good micro-scopical practice and can lead to very uneven illumination as the filament orarc will be superimposed onto the image of the specimen and subsequently,has to be subtracted digitally by mottle subtraction. Critical illuminationmight be useful, however, in those cases where the illuminating light has beenmade extremely homogeneous by light scrambling with a light fibre device(2,26,31).

Generally, it is necessary in video microscopy to provide enough light fornear-saturation of the video camera prior to applying analogue or digitalenhancement. Manufacturers of suitable equipment have red and green controllamps built into their camera control units to indicate this (e.g. HamamatsuPhotonics).

If there is insufficient light, the following measures are recommended:

(a) Use brighter lamp types (mercury or xenon lamp).(b) Redo the illumination adjustments such as setting Kohler illumination

and centring the filament or arc, possibly while observing the image on themonitor to improve.

(c) Remove ground glass diffusers from the light path.(d) Make sure that the video exit port receives 100% of the light.(e) Reduce magnification slightly.(f) Replace the otherwise highly recommended narrow band pass filter by a

wider one.(g) In AVEC microscopy near-saturation should be reached at a retardation

setting of about one-ninth of a wave (20°), as this provides the best resolu-tion (25). Further opening of the crossed polarizers far beyond 20° willrarely improve the image further, but it may introduce amounts of stray-light no longer manageable by offset and it will lead to a bright-field typeof image.

In the case of excessive light reaching the camera the following steps arerecommended:

(a) Apply weak neutral density grey filters.(b) Employ high power light sources that can be attenuated, e.g. the Atto-

Arc system (Zeiss) or metal halide burners (e.g. from Nikon).(c) Increase magnification slightly.(d) Reducing light intensity by closing the aperture diaphragm is not recom-

mended as it reduces resolution.

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(e) AVEC microscopy: if the one-ninth of a wavelength setting yields toomuch light for the camera, this could be reduced by setting compensatoror Wollaston prism to a position of less than 20°. Although widely used,this does somewhat compromise image quality especially if settings of lessthan 15° or 10° are used (3, 4, 25).

2.3.3 Full numerical aperture for highest resolution: step 3Open the condenser diaphragm fully in order to utilize the highest possiblenumerical aperture to obtain highest resolution. Also any iris diaphragm ofthe objective should be fully opened. Be careful to protect the camera fromhigh light intensity prior to this step. The result of opening the condenserdiaphragm will usually be that the optical image worsens because it becomestoo bright and flat for the eye. This setting will result in a small depth of focus,especially with DIC (optical sections of 0.3 (um or less with x 100, NA 1.4 oilobjectives). If the collection of specimen information from a larger depth offocus is desired and resolution can be sacrificed (e.g. when viewing dilutesuspensions of organelles or bacteria), the condenser diaphragm may beclosed down as desired.

2.3.4 Setting the compensator: step 4 (polarized light techniquesonly)

Set the compensator or polarizer (AVEC-POL) or the main prism (AVEC-DIC) to about 1/9 y. The optical image, that is the one seen in the oculars, willdisappear due to excessive straylight. If you have the accessories for de Senar-mont compensation as recommended by Allen et al. (3, 4) (Figure 12) this isdone by setting them at 20° off extinction. The basic set-up of de Senarmontcompensation is done as follows:

(a) Remove both Wollaston prisms and 1/4 wave plate from the light path.(b) Set analyser and polarizer to the maximal extinction.(c) Insert a 1/4 wave plate at 0° (maximal extinction).(d) Insert Wollaston prisms and set the adjustable one to the best symmetrical

extinction (if possible, check with a phase telescope for symmetry of thepattern in the back focal plane).

(e) Use the rotatable analyser as compensator and set it as desired (1/9 of awave is 20°).

If you do not have such a calibrated system, first determine the distancebetween extinction (0°) and maximum brightness (90° or 1/2 X.) by moving theadjustable polarizer, then estimate and select the 1/9 of a wave or 20° position(Figure 13). If this is not possible, one can try to find empirically a suitablesetting by shifting the movable Wollaston prism away from extinction. Manymicroscopes equipped with DIC for biological applications do not allow a

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phase shift of 90° and some may not even allow 20° since for observation by eyephase shifts of a few degrees yield good contrast. Microscope manufacturerswill, however, have the proper parts in their mineralogical programmes.

At this point you have again to make sure that the camera is protected fromexcessive light but receives enough light to work near its saturation end. Ideallythis should be near the 20° setting. Light adjustment should be done as explainedfor step 2.

In IVEC microscopy straylight is not admitted, that is the polarizers stayclose to extinction and the special rectifying optics further reduce the stray-light. Filters to reduce brightness will not be required, but much brighterlamps will most probably be necessary to saturate the camera.

2.3.5 Analogue enhancement: step 5First, increase the gain on the camera to obtain good contrast. Then apply off-set (pedestal). Always stop before you lose parts of the image that becometoo dark or too bright. Repeat this procedure several times, if necessary andhelpful. Make sure that the monitor for watching the changes is not set to ex-treme contrast or brightness, and is terminated properly (75 O) (see Section5.1.6). Analogue enhancement improves the image contrast of the specimenbut unfortunately also emphasizes dust particles, uneven illumination, and opti-cal imperfections. These artefacts, called 'mottle', are superimposed on theimage of the specimen and may in some cases totally obscure it (Figures 3 and6). Disturbing contributions from fixed pattern noise (mottle) or excessivedegrees of uneven illumination can be tolerated if digital enhancement is per-formed later (Figure 3).

If digital processing is not possible stop enhancement just before the mottleor uneven illumination becomes annoying. Apply analogue shading correc-tion and other types of analogue image improvement if your camera controlunit offers these features (see Section 1.4.1 and Figure 8). Optimal adjustmentof the lamp and thorough cleaning of the inner optical surfaces of the micro-scope, especially the surfaces in the projecting system to the camera (ocular,camera lens), usually results in images which allow the application ofconsiderably higher analogue contrast enhancement.

Finding dust. When the imaged dust particles or the mottle pattern rotatewhen the camera head is rotated, they are in the optical path before thecamera, while immobile dust is to be found on the camera target. Rotate theocular or camera objective lens to find dust located there. Dust should beremoved with a low-pressure air gun or an optical cleaning brush. If this doesnot help use lens paper or a fat-free cotton tip applicator (wooden stick, notplastic) with ethanol or ether (in the fume-hood only!). Work from the centreto the periphery in a circular fashion while carefully avoiding to apply anypressure. Dust or mottle that is defocused together with the specimen is partof the specimen.

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2.3.6 Find background scene: step 6Try removing the specimen laterally out of the field of view or (when usingDIC) defocus to render it just invisible (preferably towards the coverglass).The result is an image containing only the imperfections of your microscopesystem (mottle pattern) (Figures 3c and 6c). This step will not be satisfactory,however, with such techniques as phase-contrast or bright-field.

2.3.7 Background (mottle) subtraction: step 7Freeze, i.e. store the mottle image, preferably averaged over several frames(e.g. 64), and subtract it from all incoming video frames.

2.3.8 Return specimen to focal plane: step 8When returning to the focal plane, you should see an absolutely even andclean image, which may, however, be weak in contrast. If there are 'missing'regions that are grey and flat, there is too much contrast in the mottle of theraw image to be subtractable properly. Reduce gain, adjust offset, and repeatthe procedure (Figures 3d, e, and 6d).

2.3.9 Digital enhancement: step 9Perform digital enhancement in a similar manner to step 5, that is alternatebetween stretching a selected range of grey levels (setting 'width') and shiftingthe image obtained up and down the scale of grey levels (setting 'level' or'lower level') until a pleasing result is found. If available on your equipment,display the grey level histogram and select the upper and lower limits whichare to be defined as bright white and saturated black, respectively. If theimage is noisy (pixel noise) go to step 10 or 11.

2.3.10 Temporal filtering, frame averaging: step 10Use an averaging function in a rolling (recursive filtering) or jumping modeover two or four frames. This will still allow the observation of movementsin your specimen, but very fast motions and noise due to pixel fluctuationswill be averaged out. Averaging over longer periods of time will filter out allundesired motion, e.g. distracting Brownian motion of small particles insuspension. The image will then contain the immobile parts of the objects,exclusively.

Please note that not all image processors are capable of performing back-ground subtraction and the rolling average function simultaneously. In thiscase, averaging generally yields the better image improvement for VIM, whilebackground subtraction is more advantageous in VEC microscopy, althoughthis should be determined experimentally. Alternatively, background sub-tracted or averaged scenes (plus empty scene for later background) can bestored on video tape or disk and then subsequently be played back into theprocessor and used for further processing.

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2.3.11 Spatial filtering: step 11A number of procedures for spatial filtering is available, which can be used toreduce noise, to enhance edges of objects, or to reduce shading. These havebeen described for the analysis of single images in Chapter 8 but some imageprocessors offer such filters working at video rate so that live sequences canbe accentuated by filtering prior to recording.

2.4 InterpretationUnlike in EM images, which truly resolve the submicroscopic objects depicted(Figure 15a), the sizes of objects seen by AVEC-DIC microscopy may notnecessarily reflect their real size. Objects smaller than the limit of resolution,that is 180-250 nm, depending on the optics and the wavelength of light used,are inflated by diffraction to the size of the resolution limit, the Airy diskdiameter. The orientation of linear objects relative to the direction of DICshear may also somewhat affect their apparent thickness and contrast, if theyare oriented at angles very close to 45° or 135" (Figure I5b). Whereas the sizeof the image does not allow a decision on whether one or several objects of asize smaller than the limit of resolution are present, the degree of contrastsometimes permits this judgement to be made, A pair of adjacent micro-

Figure 15. Schematic representation of visualization of differently sized cellular compo-nents of a cytoplasm extract sample using various kinds of microscopy. (a) In transmis-sion electron microscopy (TEM) all membraneous and cytoskeletal elements arevisualized and represented in their true shapes and size relationships, i.e. they areresolved. TEM can, however, only be used with fixed (physically or chemically) material.(b) AVEC-DIC microscopy permits visualization of objects smaller than the limit of resolu-tion of conventional light microscopy but larger than 20 nm. Many objects of 20-200 nmcan be detected. These objects would, however, not appear at their real size, but inflatedby diffraction to about the size of the resolution limit (~ 200 nm). Linear objects such asmicrotutaules may lose their shadowcast appearance at orientations close to 45" or 135°(c) In conventional differential interference contrast (DIC) microscopy objects appear alsoin their typical shadowcast manner. The smallest objects visible are of an apparent size inthe order of the theoretical limit of resolution: M, mitochondrion larger than 500 nm; MT,microtubule of 25 nm in diameter; NF, neurofllaments of 10 nm in diameter; sV, axoplas-mic or synaptic vesicles of about 50 nm in diameter; V, vesicles larger than 200 nm.

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tubules would, for example, appear to have the same thickness as a single one,but the contrast would be about twice as high. If large numbers of subresolu-tion objects are crowded together and separated by distances less than 200 nmfrom one another (e.g. vesicles in a synaptic nerve ending), they will remaininvisible, because of the overlap of their Airy disk images. However, they willbe clearly visualized, if they were separated by more than the resolution limit.Also remember that, if in-focus subtraction (Figure 7) or averaging over timehas been used to create the image, all immobile or all moving parts of thespecimen, respectively, will be completely missing in recorded video scenes.

2.5 Typical applications and limitations2.5.1 Bright-field microscopyi. Advantages. Low intrinsic contrast due to low concentrations of naturalchromophores or especially vital stains can be greatly enhanced by VECmicroscopy. This can be of great advantage, since toxicity of a dye is often lessof a problem at lower concentrations. Observation in monochromatic light atthe wavelength of maximum dye absorption further increases contrast. Byusing appropriate cameras with quartz windows, even UV microscopy whichyields high resolution and high contrast images (32) is feasible to some extent,because the reduction in illumination light intensity allows a considerablylonger survival of the specimen and longer observation time.

Colloidal gold, especially when coupled to antibodies, is an important toolin immunocytochemistry at the EM level (33). Colloidal gold in sizes down to5-10 nm can also be detected using bright-field VEC microscopy (nanovidmicroscopy, see Chapter 12). Different sizes of colloidal gold will appear thesame size (0.1-0.2 um depending on focus), but occupy different grey levels.By pseudocolour conversion of these grey levels, they can, however, be differ-entiated into size classes. Thus, double labelling with different sizes ofcolloidal gold is feasible (see also Section 2.5.7).

ii. Problems with enhanced bright-field. Phase objects exhibit minimal contrastin-focus, and show opposite contrast above and below focus. It is, therefore,often very difficult to store the out-of-focus mottle image without contribu-tions from the specimen. This limits the use of high analogue contrastenhancement in bright-field microscopy.

2.5.2 Dark-field microscopyDark-field microscopy uses the light scattered from obliquely illuminatedobjects which then behave as self-luminous objects. These have been visual-ized, but not truly resolved, down to sizes of a few nanometres, if sufficientlypowerful illumination was used (up to 1000 W mercury lamps) (see Figure 2)even without video contrast enhancement.

i. Advantages. Dark-field images provide very high contrast, and this can befurther enhanced by applying gain to the video signal. Genuine straylight

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from nearby or out-of-focus scatterers can be subtracted by using offset to alimited extent, thereby increasing contrast and visibility of weak objects nextto larger ones. It may be noted that dark-field images can also be subjected tovideo-intensified microscopy.

ii. Disadvantages. The specimen should be thin and contain relatively fewscatterers. The most annoying out-of-focus scatterers do not produce truestraylight because the scattered light is not evenly distributed over the image.Because for transmitted light dark-field microscopy the objective numericalaperture must be limited, dark-field is not a very high resolution method.Nevertheless, it can detect the presence of structures in the nanometre rangein thin preparations. In epi-illumination dark-field mode it is possible, how-ever, to work at maximum numerical aperture and resolution.

2.5.3 Anaxial illuminationIn anaxial (or oblique) illumination methods the condenser aperture is un-evenly illuminated to produce a differential (shadowcast) image of increasedtrue resolution. In Abbe's original method, the condenser diaphragm ismoved to one side of the front focal plane of the condenser. In Hoffman'smodulation contrast method (34) (Modulation Optics, Inc.) undiffracted lightis partially excluded by a trizonal plate in the condenser, which also serves toaccentuate high spatial frequencies. The optional rotatable polarizer makes itpossible to vary the eccentricity of the undiffracted light. The objective singlesideband imaging technique (31) also yields very good results with VECmicroscopy. It is also possible, using a tungsten filament lamp with a hemi-spherical mirror, to displace the mirror to illuminate only half of the aperture(28). For video-enhanced contrast microscopy, these high resolution methodswork very well, because their former limitation of providing low contrastimages can be overcome by contrast enhancement.

i. Advantages. Phase details are observed in directionally shadowed differ-ential images (similar to Nomarski-DIC). High specimen birefringence doesnot interfere as in DIC microscopy. For example, myelinated vertebrate axonscan be better seen with enhanced anaxial illumination than with DIC, becausetheir highly birefringent myelin sheaths create excessive contrast in DIC. Theequipment is cheap, especially when only a bright-field microscope is required.It has been reported that observation in deeper layers of tissue is still satisfac-tory where the quality of DIC images would be much poorer (28). In prin-ciple, anaxial illumination does not reduce working aperture or resolution aslong as more than half the entrance pupil is illuminated, but can increase trueresolution, if higher order maxima of the specimen's diffraction pattern arecaptured.

ii. Disadvantages. Anaxial illumination is sometimes less sensitive thanNomarski-DIC, and the resulting image may have some anisotropy (for test

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images see ref. 36). The optical sectioning is also thicker and less precise thanwith DIC.

2.5.4 Phase-contrastPhase-contrast is an adequate method for viewing very thin, unstained speci-mens and very small phase objects, especially living cells. Video contrastenhancement is often of great value, especially in thin specimens and ifanalogue enhancement is sufficient. Enhancement is limited to cases wherethe bright halos around objects are negligible, that is with the use of thehighest magnification objectives only.

i. Disadvantage. Thick specimens imaged in phase-contrast cannot be opticallysectioned, because each image plane contains opposite contrast informationcontributed from details in the levels above and below the focus, and becausehalos are seen around phase details. Enhancement rapidly raises a very dis-turbing mottle pattern. It can sometimes be subtracted from a plane approxi-mately 0.25 um above or below the plane of interest, especially, if an area freeof specimen is used. In most cases out-of-focus mottle images of the specimenfor background subtraction cannot be obtained due to the strong contribut-ions from the out-of-focus levels of the specimen. Even extreme cleaning ofthe projecting optics is usually not sufficient to remove all the annoying mottlepattern.

2.5.5 Polarization microscopyFor visual observations of most biological objects that are only weakly bire-fringent (spindles, organelles, cytoplasm), the microscope equipped with simpleplastic polarizers is rather inadequate. The image is flooded with straylightowing to a combination of depolarization at lens surfaces, lower extinction ofthe polarizers, and strain birefringence in one or more lens elements (Table 1).

For sensitive visualization or photomicrography of weakly birefringementstructures, it was previously only possible to use a polarizing microscope witha rectifier (Nikon) that eliminated not only straylight due to depolarization atlens surfaces, but also the disturbing diffraction anomaly that can lead tospurious contrast and resolution (4, 37). The rectifier consists of a zero powermeniscus lens and a properly orientated half-wave plate (2, 24).

Using video-enhanced contrast polarization microscopy (VEC-POL), how-ever, relatively sensitive observations can be made with a simple polarizingmicroscope even if the lenses are slightly strained. One can, in fact, in mostinstances convert a bright-field microscope into a video equivalent of a rel-atively high extinction microscope by adding inexpensive film polarizers (e.g.Polaroid HN 22 polarizing material which is available in sheets and can be cutdown to any desired size; Polaroid, Inc.). In an analogy to what has been saidconcerning AVEC-DIC (see Section 2.1 and Figure 13), at 1/9 X. bias retarda-tion (AVEC-POL), the anomalous clover leaf Airy disk diffraction pattern

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normally seen at extinction (zero retardation and crossed polars) is convertedinto a normal Airy disk, resulting in the absence of spurious resolution or con-trast. In addition, the diffraction anomaly due to weak strain birefringence inany of the lens components also disappears (3, 4).

The use of the highest quality optical components for polarized lightmicroscopy together with video enhancement (AVEC-POL and IVEC-POL,see Section 2.1) allows visualization of extremely weak birefringent objects,such as individual microtubules, bacterial flagella, etc. (2, 36).

2.5.6 Differential interference contrast microscopyIn DIC microscopy the illuminating beam of plane-polarized light is split bythe first Wollaston prism into two parallel beams of polarized light passingthrough the specimen oriented at right angles to each other and very closetogether. The separation (or shear) needs to be smaller than the limit of reso-lution to prevent the appearance of double images but large enough to createa satisfying shadowing effect. Sets of Wollaston prisms with very high shear,e.g. a X 100 objective and a 200-250 nm shear, create superb contrast for visualobservation (such as some older lines from Zeiss Jena, e.g. JENAVAL), butat the increased true resolution of VEC microscopy one sees very annoyingdouble images. Generally, all medium shear pairs of Wollaston prisms aresuitable for VEC-DIC and AVEC-DIC. However, Nikon offers a special'short shear' pair of Wollaston prisms with their Eclipse series, that areespecially suitable for VEC microscopy.

i. Advantage. Differential interference contrast is the optical technique, whichis, in most applications, best suited for video contrast enhancement. TheNomarski-DIC method gives in-focus, high contrast, shadowcast images ofphase details in which the detection of shadowing is opposite for phaseadvancing and retarding details (37). The generation of contrast at high work-ing aperture is limited to a very thin depth of field, with the result that thistechnique is unique in its ability to render high contrast optical sections of only0.3 um in thickness; i.e. thinner than those obtainable by confocal microscopy(Chapter 2). Amplitude contrast can also be obtained (37). With the AVEC-or IVEC-DIC method the sensitivity of detection is increased to the level thattransparent phase objects as small as 15-20 nm can be detected under optimalconditions.

Increasing the bias retardation (Figure 13) causes a disproportionateincrease in the contrast due to small phase retardations in comparison to largeretardations. For example, if low contrast details of cytoplasmic organelles areobscured by bright structures, such as, for example starch grains, the contrastdue to the latter can be diminished by using a dimmer light source andincreasing the bias retardation more towards 1/4 A. (Figure 13).

ii. Disadvantage. Perhaps the only disadvantage of this method is that contrastdue to refraction in some highly birefringent objects (e.g. striated muscle or

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myelinated axons) can be masked by the contrast due to birefringence. In thiscase anaxial illumination is recommended as a substitute. Because in DICcontrast is directional, the specimen should be mounted on a rotatable stageand examined at more than one orientation to avoid missing weak linearfeatures aligned parallel to the direction of shear (see Figure 15). Note thatDIC microscopy uses polarized light and any birefringent material other thanthe object will show up bright at certain orientations. Strain birefringence inthe optics, in lens cement or any plastic material, makes DIC microscopyimpossible. Therefore, optic elements certified 'DIC' or 'POL' are required.Working on plastic slides or plastic Petri dishes with their unavoidable bire-fringence is impossible. If working with these is unavoidable, Hoffman contrastor anaxial illumination contrast need to be used.

2.5.7 Reflection interference contrast microscopySurface reflection interference from epi-illuminated specimens is used to imagezones of cellular attachment to glass (38). To reduce the inevitable straylightcaused by reflections from lens elements, the use of antiflex lenses designedfor metallurgical microscopes containing a 1/4 wave plate on the front surfaceof the objective was introduced. Using video enhancement, simple epireflec-tion microscopy with a 50% mirror cube but without polarizing elements orspecial antiflex lenses can be performed for several purposes.

(a) To enhance the observation of focal contacts of cell attachment to glass.(Surface reflection interference at high illumination aperture.)

(b) To enhance the observation of contour-mapping interference fringes todocument the shapes and heights of attached cells or the topology ofsurfaces of cells above a flat glass surface. (Surface reflection interferenceat low illuminating aperture.)

(c) To render visible colloidal gold or other metals down to a diameter of5 run as bright objects. Epipolarization even further improves the visibility(39, 40).

In these applications the use of antiflex objectives with 1/4 X plate (CarlZeiss GmbH) as in the original technique (38) will further improve imagequality. For further applications see Chapter 7.

2.5.8 Fluorescence microscopyWith fluorescence optics images are produced that are characterized by rel-atively high contrast and very low intensity. Therefore, fluorescence imagesusually need intensification, especially if the level of illumination has to bekept low to delay bleaching. Any straylight arising from residual autofluor-escence in lenses or the mounting medium can be removed by offset. How-ever, light from out-of-focus fluorescent structures usually cannot be treatedas straylight because of its non-random distribution. Often fluorescent details

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can be seen better when their contrast has been reversed by inverting thetelevision signal (negative image).

Fluorescent specimens have usually to be viewed with VIM but the use ofmost types of low light cameras will inevitably lead to reduced resolutionwhen compared to images obtained by VEC microscopy at the same magnifi-cation. With some applications, especially in the case of strong fluorescence itmay pay to try the use of a high resolution, high sensitivity camera (Newviconor Ultricon) in the rolling average mode (over one or more seconds). Similarlyaccumulation of images for a few seconds may bring about a useful image.

In fluorescence microscopy self-luminous objects are depicted, images con-tain, therefore, a strong contribution from out-of-focus objects. Images can,however, be limited to a depth of focus of about 1 um by digital blur-deconvo-lution (41, 42), using suitable software packages or to a depth of focus of about0.6 nm by the use of confocal microscopes (see Chapter 2). Suitable softwarepackages are provided by, for example, Bitplane AG, Carl Zeiss GmbH,ImprovisionTechnology, Inc., Inovision Corp., Scanalytics, Inc., VayTec, Inc.,or Vital Images.

2.5.9 Examples of biological and biochemical applicationsSpecimens which are extremely weak in contrast or even invisible by conven-tional microscopy are best suited for AVEC-DIC microscopy. Examples inthis class are micelles, liposomes and single or double layer membraneousmaterial, colloids (43), live, actively transcribing rDNA genes (44), synapticand other small cytoplasmic vesicles (8, 9), artificial latex particles of 50 nmand smaller, and cytoskeletal elements such as microtubules (9, 36) and actinbundles (21, 45). The process of microtubule gliding was discovered byAVEC-DIC microscopy (8), its ATP-dependence and the motor enzymeswere discovered (46) using video-microscopic motility assays (8), and evenmolecular events such as microtubule subunit assembly and disassembly canbe measured (47). Stained material or other objects, which already have highcontrast are less well suited. An overview of what can be seen and analysed bythis method in the living cell has been published elsewhere (30).

The AVEC-POL technique can visualize very weakly birefringent objectssuch as individual microtubules (36). When applied to bright-field or epipolar-ization microscopy, the VEC technique visualizes 5-20 nm diameter colloidalgold particles (39, 40) and it can be used to screen gold labelled EM speci-mens quickly in the light microscope. The same is true for semi-thin and thin,unstained, plastic-embedded EM sections (Figure 3).

Techniques to improve images of moving objects can be generated with theaid of digital processors. The 'trace' operation adds frames at predeterminedintervals to the frame memory thereby generating images showing multiplepositions of moving objects. Jumping averaging over several seconds visual-izes processes too slow to be detected otherwise, such as cell growth, chromo-some movements, and cell locomotion. Rolling averaging can be used as a

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filter to remove velocities greater than a certain pre-selected velocity from theimages. Conversely, subtraction of sequential in-focus images and scenes canbe used to view moving objects only, while stationary ones are absent fromthe image (Figure 7). The interval subtraction mode (see Section 1.3.4.v) canbe used to de-emphasize especially the slowly moving objects. If the intervalbefore the next background image is grabbed is made longer, slowermovements, which are otherwise excluded, will become part of the image.

3. Low light: video-intensified microscopy3.1 IntroductionThis technique requires the especially sensitive cameras such as cooled CCDcameras or the image intensifier units mentioned in Section 1.4.2. Once theimage has been analogue enhanced and is picked up then the digital techniquesare used as in VEC microscopy (see Section 2.3). First and most importantly,they improve the S/N during image acquisition by, for example integration,averaging, or digital filtering. Secondly, they remove any 'fixed pattern noise'such as mottle, uneven illumination, background fluorescence, or any patternemanating from the camera or the digitizing process (background/mottle sub-traction). Thirdly, they can be used for digital contrast manipulations, whileenhancement or suppression of motion is the fourth feature of VIM. Themajor difference relative to VEC microscopy in the application of these func-tions to VIM is that generally only static or slowly moving specimens can beexamined. These require the processing described in Sections 3.2.2 and 3.2.4,while a dynamic specimen (see Section 3.2.3) can be treated similarly to thoseviewed by VEC microscopy.

3.2 Procedure for image generation3.2.1 Microscope considerationsThere are no special or unconventional adjustments of the microscope neces-sary to perform VIM. One should, of course, adhere to good microscopicpractices such as cleanliness of the optics, careful alignment of the illuminationsystem, and of the camera to the light path. There are, however, a number ofpractical issues which will help ensure the best results when performing VIM.

(a) If using a camera with extended red-sensitivity (e.g. multialkali photo-cathode) for viewing in visible wavelengths it is suggested to use at leastone high quality IR cut-off filter in the camera's light path. While thehuman eye and photographic film are not sensitive to these longer wave-lengths, they can seriously degrade image quality. This is true even ifgood fluorescence filters are in place since these are notorious for passingthrough light over 700 nm.

(b) The camera image should be as parfocal with the eyepieces as possible.

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When working with very low intensity images it can be very difficult toaccurately assess exact focus from the live image on the monitor.

(c) Provisions should be made for working in a dark-room or establishingsome method to shield the objective lenses from all extraneous light.Highly intensified cameras easily pick up this light and image quality willconsequently suffer. It is necessary to cover the eyepiece of the micro-scope with, for example a black cloth or slide the shutter provided bysome manufacturers to the in-position.

(d) The illuminator should be equipped with a regulated power supply if lowintensity illumination will be utilized (e.g. to reduce photobleaching influorescence) or if quantitative measurements are planned. Small fluctua-tions in line current are greatly magnified by intensified cameras so thatstabilized power supplies are recommended.

(e) It is advisable to equip the microscope in such a way that a good opticalimage (bright-field or DIC) can be obtained for positioning and focusingthe sample prior to directing the low intensity light to the camera. Forapplications in fluorescence it is important to work with non-excitatorylight before moving into the fluorescence mode to minimize photobleach-ing effects.

(f) Since integration or averaging may be required over relatively longperiods of time, the microscope should be as stable as possible to preventany image blurring. Additionally, because many low light cameras are rel-atively large, it is often good practice to stabilize the camera by somemeans in addition to the C-mount.

(g) Care must be taken to obtain slides, coverglass, immersion liquids, etc.that are free of, or extremely low in, autofluorescence.

(h) The microscope should be equipped with a series of neutral density filters.These are used to reduce illumination of the specimen as well as toprotect the camera from excessive light.

3.2.2 Acquisition of static images (video cameras)(a) Focus the specimen through the eyepiece ensuring that no light is directed

to the camera.(b) With the camera sensitivity reduced to its minimum setting, direct the

light to the camera. If light to the camera is excessive immediately directthe light away from the camera or block the illumination path. It will benecessary to insert neutral density filters into the light path before tryingagain.

(c) With light to the camera and an image on the monitor, focus the speci-men, if necessary, and increase camera sensitivity until some portion ofthe image begins to saturate. Now reduce sensitivity to just eliminate anysaturation. This procedure ensures that the video signal is of full amplitude

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(1V peak-to-peak) and will provide the best S/N. An alternative approachto this is to increase the light level rather than the sensitivity. Obviously,this is not possible in those cases where light is limiting or where light levelsare intentionally kept low in order to reduce photobleaching, phototoxicity,phototropic effects, etc.

(d) Adjust the monitor for best picture quality. If the image is to be recorded,intermediate settings for contrast and brightness are strongly recom-mended.

(e) If the camera is equipped with gain and offset, adjust these parameters forthe best overall image quality or until that part of the specimen which isof interest displays the most information (see Section 2.3.5).

(f) Integrate the image for a suitable number of frames. Depending on thequality of the 'raw' or analogue image this can vary anywhere from 2 to512 frames. The value chosen should be based on the user's qualitativejudgement or some established criteria such as S/N ratio. As S/N is pro-portional to the square root of the number of frames, a point of diminish-ing returns is reached between 128-256 frames. It should be pointed outthat the maximum number of frames integrated may be restricted by the'bit depth' of the digital image memory. Systems for low light imagingshould be equipped with memories at least 12-bits and preferably 16-bitsdeep to allow for extended integration.

(g) Establish and maintain a background image. Two possibilities exist forthis background image. The best is to find a suitable background areaadjacent to the specimen. This area will contain all the components of theimage which should ideally be removed. This includes any shading con-tributions or fixed pattern noise of the camera, illumination irregularities,optical defects, autofluorescence in the system, and any digital noise. Ifsuch an area is not available, the second alternative is to block light to thecamera and use this 'dark image' as the background. This will correct forany camera-related phenomena. In almost no case should an out-of-focus image of the specimen be used as the background image. With fewexceptions (e.g. DIC) defocusing cannot eliminate all specimen-basedimage information and one runs the risk of erroneously subtractingnon-background information.

(h) Now, with all settings identical (camera sensitivity, illumination, etc.)integrate a background image into a different digital memory than that ofthe specimen and subtract it from the stored specimen image analogouslyto Section 2.3.7. Continue with Sections 2.3.9 to 2.3.11 to manipulatecontrast for the most pleasing or informational image.

3.2.3 Acquisition of dynamic images (video cameras)(a-e) Proceed as in Section 3.2.2.(f) Acquire a background image as in Section 3.2.2(g). Ideally, this background

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image should be integrated or averaged for the same period as the rollingaverage utilized in the next step. This image should be placed in a mem-ory which can be subtracted from each incoming video frame as in VECmicroscopy.

(g) Return to the specimen image and begin video rate background subtractionas in Section 2.3.7. If background is high, this subtraction process mayresult in the image losing intensity. If so, add digital offset to the image torestore intensity or brightness.

(h) Apply a 'rolling' or 'exponentially weighted moving' average to the back-ground subtracted image as in Section 2.3.10. This type of average offerssimilar S/N improvement [S/N = ±(2n - 1); n = number of frames] asintegration, which is used for static scenes (see Section 3.2.2, step f), butoffers the advantage of being applicable to dynamic specimens. Addition-ally, unlike image integration, in averaging the image is normalized,therefore the digital memory does not overflow or saturate.

(i) Digitally improve image contrast as in Sections 2.3.9 and 2.3.11.

3.2.4 The use of cooled, slow-scan CCD cameras to record imagesSIT and cooled CCD cameras allow the recording of signals at very low lightlevel. With a SIT camera low light signals can be recorded at video rate there-by allowing the detection of dynamic changes in real time. However, highsensitivity and high spatial resolution tend to be mutually exclusive features ofthese cameras due to signal noise. In contrast, cooled CCD cameras providehigh sensitivity while maintaining a high spatial resolution. To ensure this,images must be recorded at a slower rate. In the following paragraphs we willdiscuss some considerations on the use of slow-scanning, cooled CCD cameras.

i. Installing a cooled CCD camera on a microscopeThe CCD camera can be linked to the microscope using the C-mount connec-tor at the camera tube. The cooled CCD camera should be turned on alwaysafter the power supply for the arc lamp (e.g. high pressure mercury lamp) hadbeen ignited to avoid damage to the CCD chip by a possible power surge. Aspart of the set-up protocol one should assure that the focal plane of thecamera is properly lined up with that of the eyepieces. The software providedwith the camera will usually have a submenu to focus the specimen onto thecamera chip. The focus mode will display only a small portion of the image ata higher refresh rate. After the focus plane of the camera has been adjustedthe mounting of the camera should be secured tightly. While the cooled CCDcameras can be operated at very short exposure times (< 1 sec), prolongedfocusing on a fluorescent sample will still cause photo damage to the sample.It is, therefore, recommended to use the eyepieces for faster selecting theappropriate focal plane rather than the monitor.

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ii. Taking imagesMost high resolution cooled CCD cameras will record images with a dynamicrange of 12-bit (4096 grey levels) instead of 8-bit (256 grey levels). The highdynamic range of these cameras allows, therefore, to record fluorescenceimages. When collecting images with a high dynamic range of grey levels oneshould, however, be aware that most image processing software packagessuch as PhotoShop (Adobe Systems, Inc.) or Paint Shop Pro (Jasc Software,Inc.) can only handle 8-bit grey level and 24-bit colour images. From advancesin computer technology more sophisticated software can be expected that willbe able to handle 12-bit grey and 36-bit colour images. Keeping in mind thelimitation of the current image processing software, particular care should betaken when choosing the proper exposure time. It is recommended to takeseveral images with different exposure times.

Hi. Obtaining colour imagesMulticolour fluorescence images can be generated from specimens stainedwith multiple fluorescent dyes. In this case the correct exposure of the individ-ual images is particularly important. First, single images for each fluorescentsignal are generated using the cooled CCD camera. It should be noted herethat the original images should always be saved uncompressed and in a fileformat (e.g. .TIF) which is universally accessible by the various types of imageprocessing software. The individual images are saved as 8-bit grey images inTIF file format. Colour images are then composed in, for example, PhotoShop(Adobe Systems, Inc.) or similar programs using the 24-bit RGB option of thesoftware. Before generating a colour image the intensity range and the levelof all images to be merged should be adjusted, so that the whole range ofintensities available for that colour is used (24-bit for all three colours) (Figure16). The colour image can then be generated by opening a new image file ofthe appropriate size (matching that of the images to be merged) in the RGBmode and copying the single B/W images in either one of the red, green, orblue image layers.

3.3 Typical applicationsThe detailed information on spectral properties and the potential applicationof most dyes for the studies mentioned below are compiled in ref. 48. Thedyes can be obtained from companies such as Eastman-Kodak Company,Sigma Chemicals, and especially from Molecular Probes, Inc. which specializesin fluorescent compounds for microscopy.

3.3.1 Fluorescent analogue cytochemistryIn fluorescent analogue cytochemistry (FAC), the molecule or organelle ofinterest is isolated, purified, fluorescently labelled, and reintroduced into theliving cell (Chapter 10). Ideally, images of these 'analogues' can reveal the

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Dieter G. Weiss, Willi Maile, Robert A. Wick, and Walter S t e f f e n

Figure 16. Combined VIM and VEC microscopy of the periphery of a neuronal precursorcell (hNT PF) in culture, (a) DIG image showing organelles and cytoplasmic extensions ofthe ce l l (Newvicon camera C24-00-07, Hamamatsu Photonics). Microtubules (b) and actinfilaments (c) were detected by immunofluorescence microscopy using a cooled slow-scan CCD camera (SenSys, Photometries Ltd.). A Nikon Diaphot 300 inverted microscopeequipped with oil immersion condenser (NA 1.4), x 100 PlanApo DIC oil objective (NA1.4), a Xenon lamp (XBO 100), and a Mercury lamp (HBO 100) were employed for acquiring the fluorescence and DIC images, respectively. (d) Colour merge of (b) and (c) per-formed in PhotoShop (Adobe Systems, Inc.) in RGB mode.

distr ibut ion and organization of the antilogous nat ive molecule or organelleand how they changc over lime. For example, the rate and polarity of theincorporation of actin and tubu l in analogues i n t o in vivo structures has beenexamined using FAC as well as the changes in the cyloplasmic dis t r ibut ion ofa va r i e ty of molecules (50). Fluorescence labels can also be introduced in to

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cells as caged compounds which can be activated locally by focusing the beamof long wavelength UV light in a particular area (49).

3.3.2 Ratio imagingRatio imaging is a powerful technique in analysing the spatial and temporaldynamics of ions, molecules, and organelles in living cells (see Chapter 6). Bygenerating the ratio or quotient of two images, this technique normalizes thecell for pathlength and accessible volume, thereby allowing for quantitativeevaluations of subcellular concentrations.

3.3.3 Fluorescence recovery after photobleaching (FRAP)Studies of translational diffusion and lateral motion in membranes havebenefited greatly from the application of video-intensified imaging. With thismethod, an area of cytoplasm or membrane containing a fluorescent probe isbleached using a laser or other strong illumination source. The rate and patternof the reappearance of fluorescence in the bleached region can be used toanalyse the contribution of isotropic diffusion, anisotropic diffusion, and bulkflow to lateral transport phenomena in living cells (51, 52).

3.3.4 Molecular imagingA number of macromolecules, including DNA and actin, have been labelledwith fluorescent groups and studied using VIM (45, 53). The visualization ofsingle DNA molecules in solution has allowed the study of changes in molecu-lar conformation. Chromatin structure in isolated nuclei and intact cells hasalso been studied. Similarly, the elastic properties of single actin filaments andtheir movement on immobilized myosin have been studied (45, 54).

3.3.5 Video microspectrofluorometryThe use of intensified cameras as an alternative to photomultiplier tubesoffers the advantages of whole image, spatially resolved spectral analysis (55).Since many of the fluorochromes utilized to monitor physiological changes inliving cells exhibit measurable changes in quantum yield, spectral shape, andspectral moments, this technique shows great promise for in vivo analyses.

3.3.6 LuminescenceThe recent commercial availability of cameras with single photon sensitivityhas made it possible to image the extremely low intensity emission associatedwith several luminescent systems. Calcium transients during fertilization havebeen visualized using aequorin luminescence (56). Another, particularly excit-ing, application of VIM in this area has involved the direct visualization of geneexpression in living cells using green fluorescent proteins or the lux operon asreporter genes. The lux gene, which codes for the enzyme luciferase, can beincorporated into cells in such a way that its transcription is controlled by thepromoter of the gene under investigation; hence, activation of the promoterresults in the simultaneous emission of light (10, 57).

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3.3.7 NeurobiologyIn neurobiology, a number of VIM applications have opened unexpected experi-mental approaches. By selecting especially non-toxic and non-metabolizabledyes mammalian nerve cells and the stability of their connections can bemonitored over time in living animals. Appropriate dyes stain, for exampleneuromuscular end plates for several months, so that the innervation ofsuperficial muscle can be reinspected during small surgery (58). Voltage-sensitive fluorescent dyes can be used at low magnification to monitor theelectrical activity of the vertebrate brain with much better spatial resolutionthan was possible with EEG analyses (59). At high magnification one yieldsinformation on membrane potentials in tissue sections and single cells (60, 61).

Brain slices are a standard preparation for neurophysiological experiments.The difficulty to visualize individual neurones in standard thick slices has beena major drawback. This problem has, however, been overcome by the use ofinfrared VEC-DIC microscopy. Neurones in slices can now be visualized ingreat details, and furthermore, neuronal processes can be patch-clamped underdirect visual control. A further development of infrared video microscopy en-ables one to visualize the spread of excitation in slices making video micro-scopy a tool for the direct investigation of neuronal function (29).

4. Image analysis: video-based techniques formeasurements in living cells

Extracting quantitative data out of microscope images or image sequenceswas a relatively tedious process before the advent of video technology. Digit-ization of an image and processing its numerical representation in a digitalimage processor has made a great number of quantitative parameters access-ible with relative ease. Video microscopy has an additional advantage thatimages are already in video format, either live, on tape, or optical disk andthus can be analysed by analogue devices (see Section 1.4.6) for determining,for example, the intensity distribution along a given line (Figure 6) or distanceinformation. Such analogue devices are, however, far less versatile thandigital image analysers.

Most digital processors for video microscopy are considered real timeimage pre-processors, that is their output is an analogue video signal destinedto a monitor or recorder. If one wants to get access to the enormous variety oftechniques for digital image analysis, usually special equipment or special PC-based software packages will be required where the analogue video signal isdirectly used as input signal. Most of the processors capable of real time videomicroscopy (see Section 1.4.5) are also able to perform most of the functionsrequired for image analysis with their own processor.

As a first level of analysis, a single frame can be taken up and analysed.

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However, since video microscopy has newly opened the field of vital micro-scopy we want to be able to analyse changes in sequences of video images.Depending on the complexity of the algorithms required to extract the de-sired features it may or may not be possible to achieve this at video rate, thatis 25 or 30 times a second. In the latter case the life images from the videomicroscope or from tape are sampled such that the image analyser grabs onlyevery second, fourth, or n-th image, extracts the desired detail, and stores theinformation for later display as a function or plot. An introduction to digitalimage processing is found in refs 14 and 62.

4.1 Spatial measurements and motion analysisIt is easy to extract such parameters as size, length, width, area, perimeter, orthe coordinates of the centre of gravity for one or more objects which can bedifferentiated according to their specific grey shade. This is achieved bysetting an upper and lower threshold (binarization of the image) and can bedone automatically and in real time by most systems. In addition to con-ventional measurements, this makes accessible the analysis of motion, such asgrowth of objects (nuclei, cells, microtubules) or movement of individualorganelles in intact cells or along free microtubules (8, 9, 39, 40, 63-65).

Motion analysis of moving organelles or of microtubule ends for analysisof subunit assembly/disassembly is performed by extracting the X and ycoordinates as follows (65, 66).

(a) Store scenes of AVEC-DIC microscopy on video tape: storage ofsequences of images in a computer memory would be possible for alimited duration (one second of 512 X 512 X 8-bit B/W images takes up6 Mbyte of memory).

(b) Play back the sequences through an X,Y-tracker or a suitable softwareprogram which detect, by thresholding, a bright object in a small user-defined and -positioned frame (region of interest).

(c) Coordinates are stored at video rate or more slowly, as desired.(d) Further analyse the time series of positional data in the personal com-

puter using specific motion analysis software (65) or general time seriesanalysis software.

(e) Plot the X coordinate against the y coordinate to obtain the trace of themoving object.

(f) Plot position against time to get the velocity function. Similarly accelerat-ion behaviour and directional changes can be displayed as functions oflocation or time.

(g) Analyse the movement for intrinsic regular features by the standard tech-niques of time series analysis, such as autocorrelation and fast Fouriertransform for oscillations, or cross-correlation for similarity of the motionof different organelles.

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(h) If the object is not easily distinguishable by contrast, but visible by eye,perform an interactive frame-by-frame analysis. To this end, use a taperecorder with a 'single frame advance' feature. From each or each n-thframe extract the coordinates manually by moving a cursor with the particleor the feature of interest (65).

Simultaneous analysis of a multitude of moving objects such as organelles,swimming micro-organisms, or sperm cells may be achieved by sophisticateddedicated equipment for motion analysis (e.g. from DataCube, Inc., HaSoTecGmbH, Inovision Corp., Mitec GmbH, Motion Analysis, Inc., or UniversalImaging Corp.).

4.2 Intensity measurementsThe intensity of each digital picture element is represented by a number, inimages digitized to 8-bit accuracy, between 0 and 255. Intensity in a micro-scope image means absorption, phase retardation, fluorescence intensity, orbirefringence, depending on the technique used. If we ensure that only one ofthese contributes to the image, we can quantify these properties in the differ-ent objects comprising the image. This is easily done by using specific fluoro-chromes in fluorescence and by applying monochromatic light in bright-field(absorption image), while the other parameters are more difficult to isolate.

It should be made clear that photometers for work with cuvettes or builtinto microscopes use photomultipliers to measure integrated light intensitiesin a given slit or diaphragm with high accuracy, typically at a resolution ofseveral thousand intensity levels, but without containing spatial information.Measuring intensities in a video image, on the other hand, although givingusually only a resolution of 256 grey levels, has the advantage of providingthis information spatially resolved over 512 X 512 pixels. Intensity in the finalvideo image is the result of many steps and cannot necessarily be assumed tostill be linear or to follow Lambert-Beer's law. It is, therefore, essential, ifabsolute quantification is intended, to calibrate the system with knownstandard samples using the same optical and electronic settings (55, 67). Thisdifficulty and the relatively low photometric resolution are the two gravedrawbacks of video techniques. Cooled CCD array cameras have someadvantages for quantification (68).

The distribution of absorbing and fluorescent compounds in biologicalsamples are accessible both in the spatial and temporal domain. Hardwareand software devices to read out the intensity of a given pixel or along a line(Figure 6) or in a region of interest are available. If video sequences are analysedin the spatial domain such measurements provide information on diffusionand transport of endogenous molecules or of compounds which have eitherbeen taken up by cells, or have been microinjected. If light is shone on thespecimen, or an area of it, this can be used to measure photobleaching orFRAP of fluorescent compounds (51, 52).

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While most measurements give only estimates of amounts of the com-pounds in the light path, it is possible in some cases to measure concentra-tions. This is if images are divided by each other, such as in ratio imagingwhich is used to determine the Ca2+ or the H+ concentration (pH) (69-71) inliving cells (see Chapter 6).

These techniques, which aim towards a 'biochemistry with the microscope',that is assaying amounts and concentrations of specific molecules and ultimatelyalso enzymes, are presently in the process of rapid development. Hundreds offluorescent markers are available for specific organelles, cells, enzymes, andmembranes, which can be used in living cells to detect and analyse specificfeatures (48) (available from, for example, Molecular Probes, Inc.). Thediscussion of the special applications for different fields of biology are beyondthe scope of this chapter so that the reader is referred to the publicationsmentioned in Section 3.3 and in ref. 30.

5. Documentation and presentation of videomicroscopy data

Only the expert use of analogue and digital video technology allows profitableworking of the video microscopy laboratory. It is the aim of this section toprovide the beginner with the necessary basic knowledge of this technology.As the images generated or improved by video microscopy cannot be seen inor photographed from the microscope directly, we have to learn how to prop-erly record and archive tapes and images and then how to obtain copies oftape, hard copies or photographs of single images, or how to edit and presentvideo sequences (video clips) to larger audiences.

Traditionally documentation and presentation of video microscopy datahad been carried out by analogue methods, either by photography or makingmovie films, by analogue video recording, analogue editing, and playback onmonitors. With advancements in digital technologies we are presently witness-ing the transition to digital techniques. Therefore, it is now possible to record,print, archive, edit, and present video sequences in addition fully digitally.While recording and archiving is still dominated by analogue techniques,editing and presenting video sequences is done today by both technologies,but all single image techniques are performed almost exclusively by digitaltechnologies.

5.1 Video recordingVideo recorders are indispensable for the storage of video-microscopicsequences. They provide a comfortable way of storing the enormous amountof information, because of their easy handling and because they offer thepossibility of recording one or two (up to six) hours on one tape. The record-ings can be played back and examined immediately without processing. Before

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purchasing video equipment one should know about the standards and formatsavailable and weigh up the advantages and disadvantages of particular systems.Furthermore, one should consider new storage devices such as the DVD (digitalvideo device), which might very well replace the standard video tape recordersin the near future.

5.1.1 Video standardsThere are several colour television standards in the world. Many of the Euro-pean countries, Australia, and many African countries use the German PAL(phase alternating line) system. France, some African, and most EasternEuropean countries use the French SECAM system (SECAM = sequentiellecouleur a memoire). The NTSC standard (NTSC = National TelevisionSystems Committee), developed in the USA, is the video standard in NorthAmerica, many countries of Middle and South America, and Japan. Completelists of the TV standards of all nations are available in video shops or in ref. 2.

The American standard differs from the European ones mainly in the scanrate. The NTSC standard displays 30 images per second; each is scanned by525 horizontal lines. Because each frame is dissected into two interlaced fields('half pictures'), one containing the odd numbered lines, one the even num-bered lines, the frequency is 30 frames/sec (f.p.s.) or 60 fields/sec. Therefore,the NTSC scan rate is defined as 525/60. The two European standards have ascan rate of 625/50, that is 25 f.p.s. scanned by 625 lines.

It is important that every part of the video equipment is compatible. Thismeans that all parts of the sets must have the same TV standard. The differentscanning rates of PAL and NTSC imply that it is usually impossible to copy aPAL recording onto NTSC equipment or vice versa. While multi-standardvideo players are common, the actual transformation, that is accepting forexample PAL format and recording it in NTSC format, can be done only witha few types of special all standard video recorders.

The terms PAL, SECAM, and NTSC characterize especially the colourmodes of the standards. However, they are fully compatible with the corre-sponding black and white (B/W) standards, namely PAL and SECAM withCCIR (50 Hz) and NTSC with EIA (60 Hz). The acquisition of colour equip-ment (monitors and recorders) is recommended, because this allows one torecord B/W sequences and false colour (pseudocolour) images (see Section5.1.7) which are often very useful in video microscopy.

5.1.2 Obtaining a correct video signalThe peak-to-peak voltage of the composite video signal is standardized to amaximum of 1 V (the image information occupies the range from 0.3-1 V andthe synchronization signal (SYNC) from 0-0.3 V). In the pulse-cross mode thefour corners of the picture and the blanked region of the frame (black) whichnormally is hidden can be displayed on a monitor (see Figure 17). If the signalis deteriorated after playing back a recorded video sequence, we are able to

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Figure 17. Demonstrating the video SYNC signal with the pulse-cross mode of a monitor,(a) Correct pulse-cross display derived from a TV camera. Due to their low voltage theSYNC pulses appear as dark stripes. Vertical on the monitor, H SYNC pulses; horizontal, VSYNC pulses, (b) Pulse-cross display of a video signal from a video tape recorder. Thevertical SYNC pulse shows a distortion, (c) (d) Photograph of a time lapse scene and thecorresponding pulse-cross display. The 'flagging' in the upper part of the picture iscaused by a jammed SYNC signal.

control and try to improve the quality of the video signal using this mode.Figure I7a and b show a correct video signal from a TV camera and one froma video tape recorder with a discordant time base for the vertical SYNCsignal. Often one can improve the signal slightly by turning the TRACKINGcontrol of the video tape recorder or sliding the SKEW lever. The SKEWcontrol adjusts tension of the tape while the TRACKING control minimizesthe tracking variances between different recorders. Altering the TRACKINGcontrol may reduce even the strong distortion (flagging) often seen in timelapse scenes in the upper part of the screen (Figure 17c and d). This may alsoimprove the image if the video level of the original tape is inadvertently morethan 1 V. Normally (especially during recording) the TRACKING control hasto be set to the 'fixed' position (automatically done in most recorders).

If these measures are not resulting in a satisfying image signal the generallyadvisable steps to improve the working of delicate electronic equipment

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should be taken, such as proper grounding, connecting all parts of the set tothe same power circuit breaker, etc.

5.1.3 Video tape formatsFor use in scientific laboratories two different video tape formats are practical.These are 1/2 inch (1/2") and 2/3 inch (2/3") tapes.

i. The 1/2 inch formatThe 1/2" format is the well-known home video standard. Cassettes of the VHSformat are not compatible with devices for Beta format, which is likewiseobtainable. The VHS format has become the 1/2" standard in the world. Theadvantage of the 1/2" format is the low price for recorders and tapes. The tape,however, is thinner and narrower than 3/4" tapes, resulting in more dropouts(small white flashing spots) and noisy jams after repeated recordings thanoccur with 3/4" or Super-VHS tapes. Most of the high quality 1/2" video taperecorders offer the possibility of assembly editing. This means that one canadd scenes consecutively in a very easy way without interference between thescenes. Some high grade recorders even offer insert editing.

A considerably unproved VHS system called Super-VHS (S-VHS) has beenestablished as a new standard. The S-VHS uses the standard VHS cassettesize but offers much better resolution. Because the quality of S-VHS surpassesthat of VHS and is comparable to the 3/4" formats, it is highly recommendedfor video microscopy.

Digital-VHS (D-VHS) is the newest type of video format available. Videotape recorders for D-VHS accept VHS and the S-VHS tapes but not viceversa. D-VHS recorders can be used to record and play NTSC as well as PALstandard. Furthermore, the D-VHS is capable of bit-stream-recording andthereby allowing recording of compressed digital data sets. If connected to aserial computer interface, the system can record computer data, audio data, aswell as video sequences.

ii. The 3/4 inch formatVideo tape recorders of the 3/4" format using U-matic cassettes were wide-spread in scientific laboratories and among other semi-professional users. TheU-matic format has now been substituted by the manufacturers by the Beta-cam system. Both the U-matic video tape recorders and the cassettes areabout five times as expensive as the 1/2" ones, while the Betacam system iseven more expensive. Their value is in the higher quality of the recordings onthe thick or, more stable tape material. This is important if the user oftenneeds still frames or search mode, because these modes put a severe strain onthe tape. The 3/4" format offers a vertical resolution of approximately 340lines instead of only the 250-300 lines of 1/2" format (BAV mode).

There is no difficulty in copying from one format to another provided thatthe two video tape recorders have the same video standard (e.g. NTSC).

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However, bear in mind that you will lose quality when copying from 3/4", S-VHS, or D-VHS onto 1/2" VHS while transferring from 1/2" onto the betterformat tapes will not gain any further resolution. While the 3/4" Betacamcassette format is in use for professional recording, the U-matic format hasbeen replaced by the S-VHS format in most video microscopy laboratories.

iii. Video tape qualityThere are very different qualities of tape material obtainable. For videomicroscopy where high density and low noise recording is required only highquality tapes should be used. In addition, there are special tapes designed forstill-frame operation, a technique very often used in video microscopy. Themaximum recording time per VHS or S-VHS cassettes is six hours but thestronger one or two hour tapes are recommended.

5.1.4 Video tape recordersVideo tape recorders used in video microscopy should have several specialfeatures. For taking photographs off the monitor a good still-frame capabilityis required. To add comments later to a video film without erasing the record-ings you need a video tape recorder with AUDIO DUB capability. Most ofthese recorders have two audio channels which can be played back separatelyor synchronously. It is also convenient to have a remote control, especially ifthe video tape recorder is out of reach.

5.1.5 Time lapse recordingVarious microscopic specimens such as, for example slow particle motions orthe progress of cell division, require time lapse recording, as they becomeapparent only after speeded up play back. A time lapse recorder may be usedin parallel with a normal speed video tape recorder so you have the samesequences both in real time and time lapse for comparison. Previously recordedreal time scenes may also be speeded up later with a time lapse recorder.

There are several time lapse video tape recorders available which aredesigned to record for up to 400 hours or more onto a regular two hour tapefor observation and surveillance applications. Note that when played back atnormal speed the recordings of most of the cheaper surveillance video taperecorders show considerable interference and noise bars so that high gradetime lapse recorders are needed. However, clear still-frames can be obtainedfrom all time lapse recorders.

Time lapse recorders use regular 1/2" or 3/4" cassettes but recorded tapescannot usually be played back on a standard speed video tape recorder of thesame format. During time lapse recording the tape moves very slowly past theincessantly rotating video head, thus straining the tape. Therefore, only newand best quality tapes should be used.

Sometimes animation control units (e.g. from EOS Electronics AV Ltd., orfrom AVT GmbH), are alternatives to time lapse recorders suited for the

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documentation of very slow movements such as cell growth, locomotion, ordivision (72). These units are connected to an edit video tape recorder andpermit recording of one or several frames at intervals of 10-999 seconds.Played back in real time such recordings show at least a X 100 time lapseeffect, but they are free of image distortions.

5.1.6 Recording video-microscopic sequencesPrior to recording it is important to check the equipment, ensuring all plugsand switches are in their proper positions. A suggestion as to how to connectthe system components is shown in Figure 1. The video tape recorder shouldreceive the signal directly from the video processor and not 'at second-hand'from a monitor. For this the signal has to pass the recorder prior to display onthe monitor in the so-called E-to-E (electronics to electronics) mode. Withsome recorders this E-to-E mode picture can only be obtained when theRECORD button of the video tape recorder is pressed.

If the monitor is the last set to receive the video signal, it has to be 'termin-ated'. This means that the 75 ft switch at the back of the monitor must be setto '75 ft' position to avoid disturbances. With monitors operating in the E-to-E mode the switch is set to 'HI-Z' position. Likewise, this must be done for allthe other pieces of equipment in line. If there is no switch 75 ft/HI-Z theVIDEO OUT connector of the last piece has to be terminated with a plugcontaining a built-in 75 ft resistance. More recent equipment is automaticallyterminated.

During image processing brightness and contrast of the image are alteredelectronically. It is strongly recommended to set the brightness and contrastcontrols of the monitor always to their standard positions. This measure willprovide scenes of the same brightness level which will fit together properlywhen they are edited or processed further.

The timer and the scale bar are added to the video image by most imageprocessors. To estimate the correct magnification, especially when photograph-ing off the monitor record the scale of an object (stage) micrometer both inhorizontal and vertical position. This is used to calibrate any scaling functionor scale bar (see Figure 6) of the processor and to adjust the horizontal andvertical axes of the monitor.

Too high a level of the video signal gives rise to snowy and noisy imageswhen scenes are replayed from the video tape recorder. It is, therefore prudentto check this signal (at the VIDEO OUT connector of the image processor orvideo tape recorder) with the aid of an oscilloscope. A video level higher than1 V will cause distortion in the bright regions of the image. The recordingshould be checked by playing back the tape. If the video signal is too high, theservice personnel should be asked to reduce the output video signal to amaximum possible value of 1 V.

Although the video recording level is controlled automatically we have toadjust the audio recording level manually on some recorders, unless they are

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equipped with an audio level limiter which minimizes audio distortion at thepeaks. It is very helpful to record vocal comments during working at themicroscope, for example such events as changes of optics, the position of thespecimen, or the focal plane (note presence of microphone in Figure 1). Ifthe video tape recorder offers two separate audio channels and the audio dub-bing feature one should use channel 2 for these comments, as one can add asecond commentary afterwards (dubbing) to the previously recorded scenesonly onto channel 1.

Recorded tapes can be protected against accidental erasure. For that purposethe red cap on the bottom of U-matic cassettes is removed, or respectively,the plastic tab at the back of 1/2" cassettes is broken off. If you later decide torecord again onto these tapes, replace the button or affix adhesive tape overthe safety tab slot. The unintentional use of erasure protected tapes is thelikely cause if the video tape recorder stops working when the RECORDmode is activated.

Some frequent mistakes, which should be avoided, are the following. It isvery annoying to the person who evaluates the images if the sequences are ofshort duration. Some scientists tend to try to improve the focus of the micro-scope continually while recording, or to shift the specimen around, incessantlyexpecting better positions. These faults become conspicuous especially whenusing a time lapse recorder. During recording one must remember that suchmistakes cannot be improved afterwards and one ought to keep in mind thedesigned application of the scenes. For example, scenes which are to be usedfor motion analysis or other evaluation should last at least five, better ten,minutes without changing focus or stage position.

5.1.7 Recording pseudocolour sequencesThe pictures obtained by the camera and fed into image processors are usuallymonochrome (B/W). For special purposes, such as, for example, to enhancethe contrast by adding colours or simply to get more aesthetical pictures,many image processors permit assignment of different colours to the differentgrey values (pseudocolour or false colour). Thus all areas of the same greyvalue are coloured equally. All generated colours are coded as a mixture ofthe three basic colours red, green, and blue (RGB) and split up into the corre-sponding colour channels. This RGB signal can be displayed at high imagequality but only on monitors with RGB input capability.

To record coloured images a composite video signal of one of the inter-national standards PAL, SECAM, or NTSC is needed. Several image pro-cessors provide only analogue RGB output. These four or five signals (R, G,B, composite SYNC, or horizontal and vertical SYNC) have to be encodedinto the composite standard colour video signal. For this purpose a colour-encoder for either PAL, SECAM, or NTSC is required. This encoder is con-nected between the RGB outputs of the image processor and the video inputof the video tape recorder. The encoder, however, requires a very regular and

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exact SYNC signal, as can only be supplied by a TV camera. It is not difficult toadd colour to original microscopic scenes coming directly from the TV camera,through the image processor, and to record these scenes after encoding theRGB signal.

To colour previously recorded sequences from a tape, the video taperecorder as input source for the image processor may not provide as regular aSYNC signal as would be required. In this case, a time base corrector (TBC)may help and is connected either to the TBC socket (if available) of the firstvideo tape recorder or to the encoder. Using a TBC is often advisable asit always provides the best possible playback picture, whether for copying,editing, mixing two signals, or additional processing.

5.1.8 Digital storage media: an outlookA whole range of digital storage devices that might well replace the video taperecorders in the future are presently being available or about to be intro-duced. Generally it should be noted that a two hour movie with sound wouldrequire in the order of 4 GByte storage, depending on the algorithm of com-pression applied. From the usefulness of the different storage media in ourfield we can say the following:

i. Hard disks and RAM of PCs hold nowadays typically 5-10 GBytes or100 MBytes respectively. This means that they are very useful as temporarystorage during accumulation of special scenes, editing and preparing timelapse sequences. Hard disks are the typical intermediate storage for slowlywriting to other mass storage devices such as CD-ROM or DVD-ROM whichare presently less suitable for direct recording. The writable CD-ROM with acapacity of 650 MByte is suitable for short video clips only, however forarchiving of up to a few thousand single frames this would be the medium ofchoice. Typical B/W images are 0.4 MByte, colour images more than 1 MByte,sometimes less, depending on the resolution and degree of compression.

ii. The digital-VHS (D-VHS) tape has similar handling properties and qualityas S-VHS, with the advantage of avoiding losses in quality during copyingprocesses. Since this technology is relatively recent only few companies pro-duce D-VHS recorders.

iii. The digital video device (DVD) system was introduced for the home enter-tainment market. One DVD-ROM disk may hold a full two hour movie. Withthe introduction of DVD writers this system can be employed to store videodemo and raw data. This may become a very useful technology when thewriting process is possible at real time rather than sequences have to be ex-ported slowly from hard disk. The earlier technology of optical disk memoryrecorders (OMDR) is now extinct because the disks were too bulk, not re-writable, and too expensive (over 200.00 EURO).

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5.2 Obtaining printouts for presentation and publicationTo obtain printouts for presentation one can choose from a whole range ofmethods. While the 35 mm camera is still occasionally used to obtain stillframes from a video sequence, digital image capturing and printing, mainlyperformed on PCs, has become the technique of choice for obtaining publica-tion quality digital images and prints. Since full documentation and hand-books come with the equipment and software for the latter techniques, it maysuffice to outline the types of hardware in use.

5.2.1 Digital printsi. Frame grabber boardsA scientific quality frame grabber is not only necessary for use with high-endCCD cameras and other imaging equipment at a resolution of 640 X 480 (768X 512 for CCIR) pixels but it is also needed to capture images from inputsources including S-VHS and RGB tapes or live video. Once the image iscaptured it can be processed with a large number of appropriate imaging pro-grams such as PhotoShop (Adobe Systems, Inc.) or Paint Shop Pro (Jasc Soft-ware, Inc.). To obtain maximum image information, brightness and contrastmust first be correctly adjusted at the level of the A/DC (digitizer) by applyingthe appropriate LUT and then optimized for presentation at the level of thedigitized image (see Section 1.3.4). Special spatial imaging filters can be em-ployed to remove background noise and to improve the signal-to-noise ratio.Manufacturers of frame grabbers include Data Translation, Inc., Fast Elec-tronic GmbH, HaSoTec GmbH, Imaging Technology, Inc., Matrox ElectronicSystems Ltd., Scion Corp., or Silicon Graphics, Inc.

ii. Video printersSome effort has to be made to obtain publication quality prints. This is achiev-able with some high-end video printers which have a built-in frame grabber.Some of these printers offer lower quality hard copies on thermographicpaper, which are sufficient for many uses such as documentation. Video print-ers (e.g. Sony Corp. or Mitsubushi Corp.) are connected to the VIDEO OUTsocket of the video tape recorder. The video signal should pass the printer inthe E-to-E mode to a control monitor. When the PRINT button is pressed theactual frame or an average of the last few frames is stored and printed within afew seconds. The print quality depends on the resolution of the printer (itsnumber of grey levels, regularly 256), and on the quality of the paper used.The advantage of such video printers is the instant availability of a hard copy.The latest video printers attain almost photographic quality and have becomeavailable also in colour mode.

iii. High quality printing from digital sourcesSeveral computer-controlled, high quality printing devices are available, e.g.

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laser printers, dye sublimation printers, and ink jet printers, all of which arenow capable of producing photo-quality printouts. Choosing among the dif-ferent printing techniques will largely depend on budget and amount of print-ing needed. Dye sublimation printers are rather expensive and have highrunning costs (up to a few EURO for one A4 page). Laser printers arealso expensive but running costs are low, while ink jet printers are cheapestbut create higher costs for the ink cartridges and high-glossy, photo qualitypaper.

iv. Digital slide makersOnce video images are digitized they can also be printed directly to slide film.Several companies provide such slide printing devices, e.g. Eastman-KodakCompany, Nikon Europe B.V., Polaroid Export Europe. Printing a slide iscarried out similarly to printing to paper. While the slide makers are optim-ized for colour slides, they can also be used to generate B/W slides either byusing a special B/W slide film or by using a B/W negative film in combinationwith a reverse B/W developer kit. In contrast to colour film full saturation inblack can only be achieved using B/W film.

5.2.2 Photographic printsIf no high quality printer is available photographs can be taken from the monitorwith a 35 mm single lens reflex camera. The camera should be equipped witha 90-100 mm lens (and possibly a close-up lens) or an appropriate macrolens. Automatic exposure is not always an advantage so a camera capableof manual exposure is appropriate. Most of the latest cameras are suppliedwith an ultra-fast photocell, which is unfortunately even affected by the run-ning scan ray, which is invisible to the human eye. Therefore the informationfrom the exposure meter fluctuates widely and pictures taken with automaticexposure become often either too dark or too bright. Exceptions are theOlympus OM-2 and OM-4 cameras which do not fix the exposure at themoment the shutter is released but measure continuously during the entireexposure (off-the-film). Likewise worth mentioning are cameras with veryfast vertical shutters as found in more recent cameras, which avoid the scanbars.

To shoot B/W pictures we need moderately fast negative film such asAgfapan 100, Kodak T-Max 100, or Ilford FP4. For colour slides and colourprints load the camera only with reversal (slide) film. This is because commer-cial laboratories will be overtaxed to print correctly the mostly very unnaturalcolours of a false colour display from negative film. Using reversal film onecan point out that the colours of the print should correspond to those on theslide. Colour reversal films for good results are for example the Fuji-Velvia,Ektachrome 64, or Agfa RSXII.

For shooting photographs it is advisable to use a high resolution monitorwith a small screen. Mount the camera on a firm tripod and adjust it at right

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angles to the screen. The level of the camera lens should correspond to thecentre of the screen. The picture in the viewfinder may show either the fullmonitor with some dark background around it or only a section of the screen.Try photographing with a shutter speed of 1/8 sec or longer. Shorter times(even 1/15 sec) can result in heterogeneous pictures because of the video scanrate.

When shooting the running image at 1/8 sec exposure time the cameraeffects a kind of averaging by integrating about four frames. This reduces thenoise but may be totally inappropriate for scenes with rapidly moving objects.In these cases it is better to shoot still-frame pictures. Pressing the PAUSEbutton of the recorder provides usually only one field, that is half the numberof video scan lines as the running picture. The consequence is an image withclearly visible scan lines. It is better to store (freeze) a whole frame (twofields) in the image processor or use tape recorders with multiple heads thatdisplay whole frames also in still mode.

5.3 Preparing and presenting video sequencesTo present video sequences to small audiences monitors or LCD projectingdevices for overhead projectors are used, while for larger audiences video anddata projectors are essential (the complicated and expensive copying to 16 mmmovie film is now obsolete). With the improved display and projector technol-ogy a whole range of analogue and digital projectors is available (e.g. fromBarco International, Sharp, or Sony Corp.) which allows the presentation ofvideo signals equally well from analogue sources (tape) or digitally from harddisk or CD-ROM of a PC or laptop.

Usually tapes recorded in the laboratory are not directly suitable for pre-sentation to an audience so that a demonstration tape or clip with all scenes insuitable sequence and of proper lengths have to be compiled. Today this isdone conveniently in a fully digital way with dedicated software packages orwith special digital video editing hardware on the PC, which is supplied withsequences from various analogue (tape) or digital sources (hard disks, CD-ROMs, Internet, etc.). However, for those who still need to do it the classical(analogue) way with two video tape recorders the following hints may behelpful.

5.3.1 Copying and editing video tapesi. Preparatory considerations for editingTo copy video sequences two recorders of the same standard (PAL, SECAM,or NTSC) are required. It is possible to copy from time lapse video taperecorders with variable recording speeds onto standard speed recorders.Before beginning to edit it is profitable to compose a script where all titles andscenes are put together consecutively. For titles schedule about five seconds,for long titles the time allowance should be sufficient to enable them to be read

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slowly. Scenes should run for periods of time not shorter than 20 seconds, onlysingular scenes may be longer than one minute, important ones can berepeated.

For the copying process try to improve the video signals as much as possible.If available, the same video tape recorder as used for the original recordingshould be used since even recorders of the same standard and format are notalways completely compatible. This is caused above all by different mechani-cal adjustment (e.g. the stretch of tape) of the different sets. Checking thequality of the video signal is described in Section 5.1.2.

In the following description the source video tape recorder with the originaltape is called video tape recorder I, the receiving recorder with the editingtape is video tape recorder II.

ii. Procedure for editing video tapesBefore copying advance all source tapes to the beginnings of the requiredscenes, so that one scene after another can be added quickly onto the secondtape. The original cassettes should be protected from accidental erasure. Toobtain the more pleasing black frames instead of noise and noisy bars on partsof tape II which will not be recorded on, one should first prepare a 'black'tape by running the tape (new or used) completely through in recordingmode. Many video tape recorders will assure a smooth transition betweenrecorded scenes when the procedure outlined in Table 3 is applied.

In STOP mode (Table 3) the last few seconds of the preceding scene will beerased, but the scenes are smoothly added without frame breakdown at thesplicing point. It is only with high grade video editing equipment possible toinsert or exchange one scene for another between sequences already recorded(i.e. insert editing) in this same way without noisy frames resulting at the endof the scene.

It may in some cases become necessary to further correct the signal fromvideo tape recorder I by using a time-base corrector (TBC) which is also use-ful when adding a time bar (from a separate time date generator or an imageprocessor), a scale bar, or other overlays to an existing video sequence duringcopying.

5.3.2 Digital video editingDue to the large volume of data video capture and playback require a highlevel of computer processing power. Even for a short video sequence thesystem has to deal with a large volume of data (15^000 images for 10 min at25 f.p.s.). Processing of full screen video sequences (640 X 480) would requirea P200 Pentium processor or better, a minimum of 64 MB RAM, and 4 GB offree hard disk space. There are several systems using different approachesavailable and we mention only one typical example. Many details and usefulhints on the production and presentation of digital movies can be found inref. 73.

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Table 3. Assembly editing of video tapes for smooth transitions between scenes

Video tape recorder I (original tape)

Locate the prospected scene or title and startthe tape (PLAY mode) about 5 sec beforebeginning of this scene.

Change the cassette or wind to the nextsequence; start the tape about 5 sec beforethe scene starts.

When tape recorder II has been switchedto STOP:Prepare the next sequence.

Video tape recorder II (editing tape)

Rewind the tape to the start.Run the tape for about 5 sec in PLAY modeas a leader.Press the PAUSE button, then the PLAYand RECORD button simultaneouslywithout releasing PAUSE.

Start the tape (PLAY) about 5 sec prior to thebeginning of the desired scene.

At the beginning of the scene to berecorded release the PAUSE button, pressit again to stop the recording.

At the end: press the STOP button andrewind the tape to check it.

Rewind the tape briefly to inspect the lastscene. A few sec before its end press thePAUSE button, then the RECORD and PLAYbutton simultaneously without releasingthe PAUSE button.

At the beginning of the scene to recordrelease the PAUSE button.

One way is to control a set of tape recorders from a PC with a specialhardware/software combination allowing to run video sequences one afteranother through the image processor, improve contrast etc., select from varioustypes of smooth or other formats of transitions between the scenes, and addthem to an editing tape (e.g. Fast Video Machine from Fast Electronic GmbH).

A second approach requires a software package such as Premiere (AdobeSystems, Inc.) with a high performance scientific quality frame grabber capableof acquiring not only single frames but live video sequences (video acquisitionboard). The software package performs digital image processing functionsinstead of single frames (as PhotoShop or related packages) on wholesequences of stored, digital images. Dedicated programs such as Premiere(Adobe Systems, Inc.) will provide all the essential tools for compiling videoclips, adding special effects, composing a complete movie film, and output it inone of a variety of different file formats.

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Several, fully digital stand-alone systems, especially semi-professional andprofessional ones, are available for sometimes forbiddingly high prices. Areasonably priced exception is for instance the Casablanca system (MSMacroSystem Computer GmbH). It is capable to acquire many scenes fromanalogue and digital video sources, store them digitally and put them to orderaccording to a prepared storyboard with the selected transition effects, addtitles and audio, and copy the result to a variety of analogue or digital tapeformats.

Other manufacturers offer partially different, interesting approaches suchas MetaMorph (Universal Imaging Corp.), EDITBOX (Quantel Ltd.), or thevarious versions of video software for Silicon Graphics computers.

AcknowledgementsThe authors are greatly indebted to the late Bob Allen (1927-1986), ShinyaInoue, and the other colleagues of the Woods Hole video microscopy com-munity for sharing their knowledge and experience, for numerous discussionsand for valuable advice.

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1836.26. Schnapp, B. J. (1986). In Methods in enzymology (ed. R. B. Vallee), Vol. 134,

p. 561. Academic Press, Orlando.27. Brenner, M. (1994). Am. Lab., April, 38.28. Kachar, B. (1985). Science, 227, 766.29. Dodt, H.-U. and Zieglgansberger, W. (1994). Trends Neurosci., 537,453.30. Weiss, D. G. and Maile, W. (1993). In Electronic light microscopy (ed. D. M.

Shotton), p. 105. Wiley-Liss, New York.31. Ellis, G. W. (1985). J. Cell Biol., 101, 83a.32. Lichtscheidl, I. and Url, G. W. (1987). Eur. J. Cell Biol., 43, 93.33. De May, J. (1983). In Immunocytochemistry (ed. J. M. Polak and S. W. Van

Noorden), p. 92. Wright-PGS, London.34. Hoffman, R. (1977). J. Microsc., 110,205.35. Ellis, G. W. (1978). In Cell reproduction (ed. E. Dirksen, D. Prescott, and C. F.

Fox), p. 465. Academic Press, New York.36. Allen, R. D. (1985). Annu. Rev. Biophys. Biophys. Chem., 14,265.37. Allen, R. D., David, G. B., and Nomarski, G. (1969). Z. Wiss. Mikr. Mikrotech.,

69,193.38. Bereiter-Hahn, J. and Vesely, P. (1989). In Cell biology: a laboratory handbook

(ed. J. E. Celis), 2nd edn, Vol. 3, p. 54. Academic Press, New York.39. De Brabander, M., Nuydens, R., Geuens, G., Moeremans, M., and De Mey, J.

(1986). Cell Motil. Cytoskel., 6,105.40. Geerts, H., De Brabander, M., Nuydens, R., Geuens, S., Moeremans, M., and De

Mey, J. (1987). Biophys. J., 52,775.41. Mathog, D., Hochstrasser, M., and Sedat, J. W. (1985). J. Microsc., 137, 241 and

253.42. Wang, Y. L. (1998). Methods Cell Biol., 56,305.43. Kachar, B., Evans, D. F., and Ninham, B. W. (1984). J. Coll. Interface Sci, 100,287.44. Spring, H. and Trendelenburg, M. (1990). J. Microsc., 158, 323.45. Sase, L, Miyata, H., Corrie, J. E., Craik, J. S., and Kinosita, K. Jr. (1995). Biophys.

J., 69,323.46. Vale, R. D., Reese, T. S., and Sheetz, M. P. (1985). Cell, 42, 39.47. Weiss, D. G., Langford, G. M., Seitz-Tutter, D., and Keller, F. (1988). Cell Motil.

Cytoskel., 10,285.48. Haugland, R. P. (1996). Handbook of fluorescent probes and research chemicals,

6th edn, p. 679. Molecular Probes, Eugene, OR.

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49. Mitchison, T. J., Sawin, K. E., and Theriot, J. A. (1998). In Cell biology: a laboratoryhandbook (ed. J. E. Celis), 2nd edn, Vol. 3, p. 127. Academic Press, New York.

50. Mittal, B., Sanger, J. M., and Sanger, J. W. (1987). J. Cell Biol, 105,1753.51. Peters, R. (1985). Trends Biochem. Sci., 10,223.52. Kapitza, H. and Jacobson, K. (1986). In Techniques for the analysis of membrane

proteins (ed. C. I. Ragon and R. J. Cherry), p. 345. Chapman and Hall, London.53. Yanagida, M., Morikawa, K., Hiraoka, Y., Matsumoto, S., Uemura, T., and Okada,

S. (1986). In Application of fluorescence in the biomedical sciences (ed. D. L. Taylor,et al.), p. 321. Alan R. Liss, New York.

54. Kron, S. J. and Spudich, J. A. (1986). Proc. Natl. Acad. Sci. USA, 83, 6272.55. Wampler, J. E. (1986). In Application of fluorescence in the biomedical sciences

(ed. D. L. Taylor, et al.), p. 301. Alan R. Liss, New York.56. Yoshimoto, Y., Iwamatsu, T., Hirano, K., and Hiramoto, Y. (1986). Dev. Growth

Differ., 28, 583.57. O'Kane, D. J., Lingle, W. L., Wampler, J. E., Legocki, M., Legocki, R. P., and

Szalay, A. A. (1988). Plant Mol Biol, 10,387.58. Purves, D. and Voyvodic, T. (1987). Trends Neurosci., 10, 398.59. Blasdel, G. G. and Salama, G. (1986). Nature, 321, 579.60. DeBiasio, R., Bright, G. R., Ernst, L. A., Waggoner, A. S., and Taylor, D. L.

(1987). J. Cell Biol, 105, 1613.61. Zecevic, D. (1996). Nature, 381,322.62. Shotton, D. (1993). In Electronic light microscopy (ed. D. Shotton), p. 39. Wiley-

Liss, New York.63. Cohn, S. A, Ingold, A. L, and Scholey, J. M. (1987). Nature, 328,160.64. Weiss, D. G., Keller, F., Gulden, J., and Maile, W. (1986). Cell Motil CytoskeL, 6,

128.65. Weiss, D. G., Galfe, G., Gulden, J., Seitz-Tutter, D., Langford, G. M., Struppler,

A., et al. (1990). In Biological motion (ed. W. Alt and G. Hoffmann). Lecture notesin biomathematics, Vol. 89, p. 95. Springer-Verlag, Berlin.

66. Soll, D. R. (1988). Cell Motil. CytoskeL, 10, 91.67. Wampler, J. E. and Kutz, K. (1989). Methods Cell Biol., 29,239.68. Shaw, P. J. (1993). In Electronic light microscopy (ed. D. M. Shotton), p. 211.

Wiley-Liss, New York.69. Tsien, R. Y. and Poenie, M. (1986). Trends Biochem. Sci., 11, 450.70. Bright, G. R., Rogowska, J., Fisher, G. W., and Taylor, D. L. (1987). BioTech-

niques, S, 556.71. Bright, G. R., Fisher, G. W., Rogowska, J., and Taylor, D. L. (1989). Methods Cell

Biol., 30,157.72. Allen, T. D. (1987). J. Microsc., 147,129.73. Waterman-Storer, C. M., Shaw, S. L., and Salmon, E. D. (1997). Trends Cell Biol.,

7, 503.

Further readingVideo microscopyRef. 2.Sluder, G. and Wolf, D. E. (ed.) (1998). Video microscopy. Methods in cell biology,

Vol. 56. Academic Press, New York.

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Shotton, D. M. (1998). In Cell biology: a laboratory handbook (ed. J. E. Celis), 2nd edn,Vol. 3, p. 73 and p. 85. Academic Press, New York.

Shotton, D. M. (ed.) (1993). Electronic light microscopy: the principles and practice ofvideo-enhanced contrast, digital intensified fluorescence and confocal laser scanningmicroscopy, p. 355. Wiley-Liss, New York.

YEC microscopyRef. 36.Weiss, D. G. (1998). In Cell biology: a laboratory handbook (ed. J. E. Celis), 2nd edn,

Vol. 3, p. 99. Academic Press, New York.VIMRefs 6,18,48, and 71.Taylor, D. L., Waggoner, A. S., Murphy, R. F., Lanni, F., and Birge, R. R. (ed.) (1986).

Applications of fluorescence in the biomedical sciences. Alan R. Liss, New York.Digital image processingRefs 14 and 62.Jahne, B. (1991). Digital image processing, p. 337. Springer-Verlag, Heidelberg.Anonymous (1985). Image analysis. Principles and practice. Published by Joyce-Loebl,

distributed by IRL Press.Baxes, G. A. (1984). Digital image processing. Prentice-Hall, Englewood Cliffs, New

York.InstrumentationRefs 2 and 23.Guide to biotechnology products and instruments. (1988). Science, 239, G73 and

G164-G180.

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Microscopy of chromosomesA. T. SUMNER and A. R. LEITCH

1. IntroductionChromosomes have been studied for something like 120 years, and for mostof this period they were visualized using uniform staining with a variety ofdyes. This was adequate for studies of the gross structure and behaviour ofchromosomes, but identification of individual chromosomes was difficult,being based only on chromosome morphology and size, and specific chromo-some substructures could not be studied. Just over 25 years ago, two new tech-niques emerged that revolutionized the study of chromosomes:

(a) Chromosome banding, a series of staining techniques that producedpatterns of longitudinal differentiation on chromosomes, and allowedidentification of specific chromosomes and chromosomal substructures.

(b) In situ hybridization, which labels specific DNA sequences in chromosomes.

Methods for chromosome banding are described in Section 4, and methodsfor in situ hybridization in Section 5. In recent years, immunocytochemistryhas been applied increasingly to studies of chromosomes; these methods aredescribed in more detail in Chapter 5. However, methods of preparingchromosomes for immunocytochemistry do differ from routine chromosomepreparation methods, and a range of preparation procedures is described inSection 2. Methods of recording images of chromosomes are of vital import-ance: fluorescent images are increasingly popular for a variety of reasons(greater sensitivity and contrast, and multiple labelling techniques), yet cansuffer from low brightness and impermanence. It is therefore not surprisingthat some of the technically most advanced equipment for image capture hasbeen applied to the study of chromosomes, although photographic film will nodoubt also continue to be important for the foreseeable future. Methods forrecording images of chromosomes are discussed in Section 6.

2. Methods of preparing chromosomesIn this section it is only possible to give a few examples of how to obtainmitotic cells, and of methods of preparing them for the different procedures

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A. T. Sumner and A. R. Leitch

described later in, this chapter. For a wider variety of procedures, includingdifferent types of mammalian tissues and a variety of plants, lower vertebrates,and invertebrates, see refs 1-6. Methods for mammalian meiotic chromosomesare described in detail by Chandley et al. (7).

There are two aspects to preparing chromosomes: obtaining growing cellswith a sufficient proportion of mitotic cells to make it practicable to studytheir chromosomes, and spreading the chromosomes on to a microscopeslide so that they can be seen easily. It is very hard work if only one cell in athousand is dividing, but it may sometimes be necessary to use such material ifit is difficult to obtain or grow. If the dividing cells are present at a few percent, that is perfectly adequate for most purposes, although by using synchro-nized cells, preparations can be obtained in which over 90% of cells are inmitosis.

2.1 Routine preparation of mammalian chromosomesIn this section, two methods of culturing mammalian cells will be described,and a single method of fixing them and spreading them on slides. Such prepa-rations are suitable for plain staining of chromosomes, to count the number ofchromosomes in the cell, to study chromosome aberrations, and to measurethe amounts of DNA in chromosomes, for chromosome banding, and for insitu hybridization. They are not generally usable for immunocytochemicalstudies.

2.1.1 Culture of human blood lymphocytesThis is a routine type of procedure used in clinical cytogenetics laboratoriesall over the world. A small blood sample is added to a suitable culturemedium. The medium contains phytohaemagglutinin, which induces thelymphocytes to grow and divide. After an appropriate period of culture(usually two to three days), colchicine or some other inhibitor of micro tubuleformation is added to arrest the cells in metaphase, and the cells can then beharvested, fixed, and spread.

Protocol 1. Human lymphocyte culture

Reagents• Culture medium: 8 ml F10 medium (Gibco • 10 ug/ml colcemid (Sigma)—keep in the

BRL), 2 ml fetal calf serum, 0.1 ml phyto- refrigerator and protect from lighthaemagglutinin (PHA), 0.01 ml antibioticmixture (10000 U/ml penicillin, 10 mg/mlstreptomycin)—keep in the refrigerator orcold room

Method1. Collect adult blood by venipuncture into a sterile container coated with

lithium heparin (anticoagulant). Mix gently to prevent clotting. Blood

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should only be taken by a qualified person, and all procedures must becarried out under sterile conditions.

2. Incubate 0.5 ml blood for 72 h at 37 °C in 5 ml culture medium.

3. 3 h before harvesting (i.e. 69 h after setting up the culture), add 0.1 mlcolcemid solution.

4. Harvest cells (see Protocol 3).

2.1.2 Chinese hamster ovary (CHO) cellsCHO cells are a continuously growing cell line. During interphase the cellsremain attached to the substratum, but become detached during mitosis. Avery high proportion of mitotic cells can therefore be obtained simply byshaking the culture vessel gently, and pouring off the culture medium, whichwill generally contain more than 90% mitotic cells.

Protocol 2. Culture of CHO (Chinese hamster ovary) cells

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Equipment and reagents• C02 incubator set at 37 °C• Inverted microscope with phase-contrast

optics• 10 (jig/ml colcernid (see Protocol 1)

• 50 ml Falcon plastic culture vessels (BectonDickinson)

• RPMI 1640 medium containing 10% fetalcalf serum (Sigma)

Method

1. Add approx. 106 CHO cells to the culture vessel, and make up to 8 mlwith the culture medium.

2. Culture for approx. 48 h in an incubator at 37°C, ventilated with an airmixture containing 5% carbon dioxide.

3. Examine the culture flask using the inverted microscope. If the cellsare nearly confluent, proceed to the next step; otherwise, return themto the incubator, and continue to examine the flasks at intervals untilthe required density of cells is attained.

4. Add 40 |xl colcemid, and culture for a further 3 h.

5. Harvest the mitotic cells by shaking the flask gently, and pouring offthe supernatant culture medium, which contains mainly metaphasecells.

6. Treat the cells as described in Protocol 3.


2.1.3 Fixation with methanol:acetic acid

Protocol 3. Fixation and spreading of routine chromosomepreparations

The same procedure is used for both cultured human lymphocytes andfor CHO cells.

Equipment and reagents• Microscope slides, preferably with frosted

ends. These can be bought already cleaned,but it is nevertheless advisable to cleanthem further by soaking them in acid:alco-hol (1% concentrated HCI in absolute alco-hol). Keep a large jar with a supply of slidesin it, and take these out and clean them justbefore you are ready to harvest the cells.Slides should be wiped dry and polishedwith muslin.

» Bench-top centrifuge, with buckets for 10ml tubes

• Vortex mixer with speed regulator• 0.075 M (hypotonic) potassium chloride

solution (5.6 g/litre)• Methanol:acetic acid fixative: 100%

methanol, 100% glacial acetic acid (3parts:1 part), prepared immediately beforeuse—discard any unused fixative

Method

1. Place the culture medium containing the cells in a 10 ml centrifugetube, and centrifuge at about 200 g for 5 min.

2. Pour off the supernatant, and add 10 ml hypotonic potassium chloridesolution to the pellet of cells, with gentle shaking on the vortex mixer.Ensure that the cells have all dispersed, and stand for 10 min at roomtemperature.

3. Centrifuge again at 200 g, and pipette off the supernatant without dis-turbing the pellet of cells.

4. Disperse the cells with the vortex mixer, and, keeping the cells mov-ing by gentle application of the vortex mixer, add the methanol:aceticacid fixative drop by drop until there is a great excess of fixative, thenmake up to approx. 10 ml.

5. Centrifuge the cells at 200 g again, and decant off the supernatant.

6. Add 10 ml fixative again, with vortexing, and centrifuge the cells asbefore.

7. Decant off the supernatant, add fixative, and centrifuge, as in step 6.

8. Decant off the supernatant and add sufficient fixative, with vortexing,to produce a slightly milky fluid. The precise appearance required atthis stage to obtain a good number of cells on the slide, with goodspreading of chromosomes, will be found by experience.

9. Take a clean microscope slide, and write on the frosted end the iden-tification of the culture, plus any other relevant details (e.g. date).

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10. Using a polythene pipette, make sure the fixed cell suspension isthoroughly dispersed, and take an aliquot up into the pipette (0.1-0.2 ml should be ample at this stage). Drop two or three drops ofthe cell suspension on to different parts of the slide from a height of5-10 cm. Breathe gently on the slide to aid spreading of the chromo-somes.

11. Allow the cells to dry, and examine the slide with a phase-contrastmicroscope for density of cells and proportion of metaphases (thiscan easily be assessed with a x 10 objective); a x 40 objective maybe used to assess the quality of spreading of the chromosomes ifnecessary.

12. If everything is satisfactory, prepare as many slides as required in thesame way.

13. The fixed cell suspension can be stored overnight in the refrigerator;for longer storage, place in a freezer at -20°C. Allow the cell suspen-sion to warm to room temperature, centrifuge, and resuspend infresh fixative, before using again.

2.2 Preparation of cells by cytocentrifugation forimmunocytochemical studies of chromosomes

Preparations fixed in alcohol:acetic acid are generally unsuitable for immuno-cytochemical studies, as the fixative extracts or denatures many proteins. Aradically different approach is therefore required to produce spreads withoutacid treatment. In general, such methods spread the chromosomes by cyto-centrifugation, in which the cells are centrifuged on to a microscope slide.Such preparations may still contain a considerable amount of cytoplasm, whichmay have to be removed to allow access of immunocytochemical reagents.Fixation is not necessary, and indeed may be undesirable, before cytocentri-fugation. Once the cells have been centrifuged on to the slide, they may befixed in the most appropriate way for the antigen being studied, as differentantigens differ in their susceptibility to destruction by fixation.

The method given here has produced satisfactory results in the first author'sexperience, but other published methods are equally suitable (8). The qualityof the chromosome morphology is unlikely to be as good as that obtainedroutinely with alcohol:acetic acid fixed preparations: some chromosomes maybe very poorly spread, especially if the cells are too concentrated on the slide,while, particularly at the edges of the preparations, others may become exces-sively stretched. In between, there should be a proportion of cells in which thequality of the chromosomes is adequate.

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Protocol 4. Cytocentrifuge preparation of chromosomes

The method described here is suitable for cells cultured as described inProtocols 1 and 2.

Equipment and reagents• Cytocentrifuge: any model appears to be

equally suitable, but the instructions givenbelow refer to a Shandon Cytospin 2 (quan-tities, centrifugation speeds, times, etc.,may have to be adjusted for other models)

• Phosphate-buffered saline (PBS): Dulbecco'A' tablets, dissolved according to the manu-facturer's instructions (Oxoid Ltd.)

. 0.1% Triton X-100 (v/v) in PBS

• Stenman's hypotonic solution (9): 10 mMHepes Na ((/V-2-hydroxyethyl) piperazine-W-2-ethanesulfonic acid monosodium salt,2.603 g/litre) pH 7, 30 mM glycerol (2.19 ml/litre), 1 mM calcium chloride (CaCI2.6H2O,0.2191 g/litre), 0.8 mM magnesium chloride(MgCI2.6H20, 0.1626 g/litre)—store in therefrigerator

Method

1. Place the culture medium containing the cells in a 10 ml centrifugetube, and centrifuge at about 200 g for 5 min.

2. Pour off the supernatant, and add 10 ml Stenman's hypotonic solutionto the pellet of cells, with gentle shaking on the vortex mixer. Ensurethat the cells have all dispersed, and stand for 10 min in the refrigera-tor at approx. 4°C.

3. Disperse the cells in the Stenman's hypotonic, and add aliquots of0.3-0.4 ml of the cell suspension to the centrifugation chambers of theShandon Cytocentrifuge.

4. Centrifuge for 10-15 min at 1500 r.p.m.

5. Remove the slides from the Cytocentrifuge, lay them flat, and allowthem to dry out.

6. Later (the same day or the next day), treat the slides with Triton X-100solution at room temperature for 5-30 min to remove cytoplasm.

7. Wash for 5 min each in three lots of PBS.

8. Fix the preparation as required, and proceed with immunocytochemi-cal labelling according to standard procedures (see Chapter 5, and alsoProtocol 9).

2.3 Preparation of chromosomes from plant cellsPlant mitotic cells can be found in the apical meristems of roots and shoots,and in dividing tissues in ovaries, anthers, and endosperm. In addition theycan be generated from tissue cultured materials. Whatever the source, it isimportant that the material is vigorous and healthy at the time of harvest. Thequality of material is an important factor in obtaining high quality chromo-some preparations.

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Root tips are the commonest source of mitotic divisions in plants. Typically,seeds are germinated on moist filter paper or tissues, or more rarely on 2%(w/v) agar overlaid with cellophane discs. In either way young, fresh, root tipscan be obtained in many species. If it is impossible to obtain roots from germi-nating seeds, then try to obtain them from cuttings or root cultures. The finaland least favourable option is root material from soil grown plants. In all casesmake sure the roots are young, vigorous, and in almost all cases with a whiteapex. Other good sources of metaphases are young anthers and ovaries;meiotic tissues can also be obtained from anthers at later stages. Howeveraccumulating metaphases in buds is more problematic than it is with roots.The final source is tissue cultured material. Long-term cultures, especially sus-pension cultures, will almost certainly have rearranged karyotypes. Howeverexperimental manipulation of growing conditions and the use of cell cycleinhibitors can lead to high metaphase indices.

2.3.1 Accumulation of metaphasesIn general metaphase indices are lower for plant material than for mammalianmaterial, although accumulation protocols have been developed which cansubstantially increase mitotic indices (10). Often it is desirable to spend timeincreasing the mitotic index of plant material to help chromosome bandingand in situ hybridization experiments. Examine vigorously growing materialfirst without any treatments. Then experiment with the protocols describedbelow, which are simple and will provide a basis for accumulating metaphases.

Excised root tips, buds, or other meristems are treated with one of thefollowing solutions. Times and conditions need to be determined empirically.When accumulating metaphases in small seeded plants, treat the whole seed-ling. For big seeded plants, cut off the root tip. 0.01-0.05% (w/v) colchicine isideal for most plants. Material should be placed in the solution for 3-6 h atroom temperature or 16-24 h at 4°C. For cereals, treatment with aerated dis-tilled water at 0°C (ice-water), typically for 24 h, is ideal. For some dicoty-ledonous plants, material can be placed in 2 mM 8-hydroxyquinoline for 1-2 hat room temperature followed by 1-2 h at 4°C.

2.3.2 Fixation and chromosome spreadingThere are two main ways to prepare plant material for chromosome studies:'squashing' and 'dropping'. Dropping involves fixing material in ethanol:acetic acid (3:1), making a cell suspension with enzymes, and droppingthe suspension on to a glass slide, much as is done for mammalian cells(Protocol 3) (1). This method gives the most sensitive in situ hybridizationdata and may be essential for detecting single and low copy DNA sequences(11). However, because there is no hypotonic step, the chromosomes tend tobe poorly separated. Squashing, on the other hand, gives good separation ofchromosomes, ideal for high quality chromosome banding, but at the cost ofsignal strength when performing in situ hybridization. The protocol below is a

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gentle squashing method which combines advantages of both methods and inour hands is successful. It is important through practice to develop a methodthat works for your material.

Get good spreads before chromosome staining, as the quality will neverimprove after the staining! Under the phase-contrast microscope and espe-cially the Nomarski differential interference microscope, cytoplasm can beseen easily. Avoid slides with too much cytoplasm overlying the cells. Chromo-somes and interphase nuclei with nucleoli should be clearly seen. Cells andnuclei from meristematic cells after squashing should be round. They shouldnot have rectangular outlines and bright walls. Such cells are usually poor andslides containing a lot of material like this should be discarded. The optimaldensity of cells is when individual cells do not overlap but are not so few thatthey are hard to find. Material with tiny chromosomes may be screened bestusing DAPI fluorescence, and slides that are selected washed in 2 X SSC(saline sodium citrate buffer, see Protocol 6) and immediately used for in situhybridization. Poor slides give poor in situ hybridization results and it is worthscreening carefully.

Protocol 5. Fixing and spreading plant chromosomes

Equipment and reagents• Acid cleaned slides (see Protocol3)• Watch glass• Hypodermic needles and forceps• Razor blade• Diamond pen• Binocular microscope, and phase-contrast

or Nomarski interference microscope• Fixative: ethanol, glacial acetic acid (3:1),

freshly prepared. 10 x enzyme buffer pH 4.8: 40 mM citric

acid, 60 mM sodium citrate—store as astock solution at 4°C

• 1 x enzyme buffer: dilute 10 x enzymebuffer with water (1:9)

• 2 x enzyme solution: 2% (w/v) cellulase(1.8% (w/v) dry powder from Aspergillusniger [Calbiochem] plus 0.2% (w/v)'Onozuka' RS [Yakutt Pharmaceutical Ind])and 10% (v/v) pectinase (Sigma) in 1 xenzyme buffer—store in aliquots at -20°C

• 45% acetic acid• 2 ug/ml DAPI (4',6-diamidino-2-phenylin-

dole) in 2 x SSC• Dry ice

Method

Vigorously growing healthy material is fixed in freshly prepared fixativefor at least 10 h at room temperature. Material can be stored for at least amonth at -20°C.

1. Place 0.5-1 cm of root tip or individual anthers/ovaries into 1 xenzyme buffer in a watch glass. Replace solution continuously over25 min period by pipetting fresh 1 x enzyme buffer into the watchglass.

2. Remove 1 x enzyme buffer with a pipette and flood material with 2 xenzyme solution. Incubate at 37°C.

3. Regularly check the state of digestion. Material should be close to

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falling apart but still integral, e.g. root tips picked up by their cut endshould not be able to support their own weight. Typical digestiontime is 30 min.

4. Remove 2 x enzyme solution and replace with enzyme buffer. Washfor 25 min with continuous replacement of 1 x enzyme buffer.

5. Replace 2 x enzyme solution with fresh fixative by removing half thevolume of 1 x enzyme buffer and replacing with fixative. Repeatthree times and then three times in fresh fixative.

6. Remove fixative and replace with 45% acetic acid.7. Under a binocular microscope excise meristematic tissues with the

needles and place in a small drop of 45% acetic acid on a glass slide.In the case of root tips the area of interest is the white zone immedi-ately behind the root cap. Do not take more tissue than is needed, asmore tissue usually means a lower quality of spreading. Break up thematerial on the slide with the hypodermic needles.

8. Carefully add a 24 x 24 mm coverslip, making sure that no airbubbles are trapped.

9. Gently tap the material and monitor its dispersal with the phase-contrast or Nomarski microscope.

10. Place a filter paper over the coverslip, blot gently, and press lightlywithout shearing the coverslip. Examine the slide again. Regulate thepressure of spreading and the amount of tapping to optimize spread-ing. In general, the harder that it is pressed, the lower the quality of insitu hybridization.

11. Place good slides on dry ice, leave for 5 min, and flick off the coverslipwith the razor blade. Leave slides to dry.

12. Select high quality slides under phase or Nomarski microscopes.

2.4 Assessment of the quality of chromosome preparationsChromosome preparation from animal cell cultures and plant meristemsremains as much an art as a science, although attempts are being made tounderstand the process better (12). It is therefore necessary to be able tojudge the quality of chromosome preparations as soon as the first slides aremade, so that appropriate adjustments can be made at once to obtain satisfac-tory spreads from the material. Occasionally, cells fail to grow vigorously, orproduce such a low mitotic index that they are virtually useless; if this occurs,seek advice from an expert in the material of interest.

For routine, alcohol:acetic acid fixed preparations, the aim is to achievemetaphase spreads in which there are few if any overlapping chromosomes,yet none have been lost, while at the same time eliminating all the surroundingcytoplasm, which is likely to interfere with subsequent staining and labellingreactions. Possible remedies for a variety of faults are described in refs 3, 5,

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A. T, Sumner and A. R. Leitch

and 13. Although it is possible to stain slides quickly to assess their quality, itis quicker to look at the unstained preparations using a phase-contrast micro-scope, or even better, a Nomarski interference microscope if one is available.The advantage of the latter is that chromosomes, cytoplasm, and cellular com-ponents are easily visualized in unstained dry spreads.

For cytocentrifuge preparations, it is important to have the right number ofcells present: if they are too concentrated, the chromosomes will not spread,and if there are too few cells, the chromosomes may tend to be overstretched.To some extent these problems can be overcome by adding less or more cellsuspension to the cytocentrifuge buckets, but it is probably better to dilute orconcentrate the cell suspension.

3. Uniform (solid) staining of chromosomesAlthough it would be impossible to carry out a comprehensive chromosomalstudy of a species without using banding methods (see Section 4), there arestill good reasons for using uniformly, non-banded chromosomes from time totime. The chromosomes do not suffer the distortion that often occurs withbanding, the morphology of the chromosomes is clearer and the ends, in par-ticular, are clearly visible, and chromosome abnormalities and fragile sites aremore easily seen. Solid staining with methods such as Feulgen (2,4) is valuablefor making DNA measurements. In addition, solid staining is valuable as acounterstain for various other banding and labelling techniques, as describedbelow. Solid staining methods can be divided into two classes: transmittedlight stains, such as aceto-Orcein, Giemsa, and Feulgen, which provide perma-nent preparations; and fluorescent stains such as DAPI, propidium iodide,and Acridine Orange, which are impermanent, but often very quick to carryout, provide good counterstains for fluorescent labelling (for in situ hybridiza-tion and irnmunocytochemistry), and can be used to measure DNA (both onslides and by flow cytometry).

Full details of these methods will not be given here. For many purposes,indeed, it is not necessary to specify a precise procedure: an appropriate volumeof water or any buffer that is to hand (e.g. phosphate-buffered saline) can be putinto the staining vessel, and a small amount of a stock dye solution added. Thechromosome preparations are stained until sufficient staining is obtained, andmay be put back for longer if necessary. For fluorochromes, the dye concentra-tion and staining time required may be surprisingly low. For the use of fluoro-chromes as counterstains to other procedures, see Protocols 9 and 12.

Giemsa staining can be done very quickly and easily, but will stain anysurrounding cytoplasm as well, a particular problem with chromosomes pre-pared by squash techniques (i.e. plant and invertebrate chromosomes). Insuch cases, it will be better to use aceto-Orcein (5) or Feulgen (2, 4) staining,although both of these methods are rather long and complicated. If a fluor-escence procedure is acceptable (i.e. permanent preparations are not required)

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DAPI is appropriate, as its blue fluorescence cannot be confused with the redautofluorescence of chloroplasts, for example.

4. Chromosome bandingChromosome banding refers to the production of longitudinal differentiationalong chromosomes by the use of special staining methods, in the absence ofany structural differentiation (14). Specific chromosomes can be identified bytheir characteristic patterns, not only within a species, but" also in relatedspecies. Some types of banding draw attention to specific parts of chromo-somes, and others are related to the functional properties of specific regions,such as nucleolar organizing regions (NORs) and kinetochores. Thus chromo-some banding can also be used to study various aspects of chromosomal func-tion. Banding patterns also draw our attention to certain aspects ofchromosome organization which are of interest in their own right (14, 15).

4.1 The classification of chromosome bandsFour different classes of chromosome bands have been recognized (14). Hetero-chromatic bands (C-bands, Section 4.2, Protocol 6) form discrete blocks onchromosomes, and are nearly always found at the centromeres, and sometimesalso at terminal and interstitial sites. They commonly (but apparently notinvariably) contain highly repeated DNA sequences, and lack conventionalgenes. Heterochromatic bands appear to contain distinctive proteins whichmay contribute to their compactness. Note that all the methods for hetero-chromatic bands stain only constitutive heterochromatin, but not facultativeheterochromatin (such as the inactive X chromosome in female mammals).

Euchromatic bands consist of a series of positively and negatively stainedbands throughout the non-heterochromatic parts of the chromosomes. Theeuchromatic bands, which can be stained by such methods as G-banding(Section 4.3, Protocol 7), R-banding, or various fluorochromes, are largelyconfined to higher vertebrates (reptiles, birds, and mammals), althoughreplication bands, which generally show patterns corresponding to those pro-duced by the aforementioned methods, may well be universal (14). It is stillnot clear whether the lack of euchromatic bands in the chromosomes ofalmost all plants, invertebrates, and lower vertebrates genuinely represents adifference in the organization of the chromosomes in these organisms, orwhether their absence is due to technical problems which have not yet beensolved. An extensive listing of the properties of euchromatic bands is given byHolmquist (16). During chromosome condensation, dark G-bands fusetogether, obliterating the pale bands between them, so that the chromosomesshow fewer bands at metaphase than at prophase. The important practicalfeature of euchromatic bands is that they form distinctive patterns character-istic of each chromosome pair of a species, and can be used to identify thatchromosome, even when translocated or rearranged.

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The NORs are those segments of the chromosomes that contain the genesfor ribosomal RNA, and on which the nucleoli are formed. The NORs, whichcontain hundreds or even thousands of copies of the ribosomal genes, com-monly appear as constrictions of the chromosomes. They can be stainedspecifically with silver (Section 4.4, Protocol 8).

The final class of band is formed by staining the kinetochores, which are thesites of attachment of the spindle microtubules to the chromosomes. The pre-ferred method for demonstrating kinetochores uses an immunocytochemicalprocedure (Section 4.5, Protocol 9).

In the following sections, one principal banding method has been describedin detail for each of the four classes of chromosome banding. There are, how-ever, a considerable number of methods that may prove useful in variouscircumstances, and although there is no space here to describe the protocols,it is as well that the reader should be aware that other techniques are avail-able. Technical details will be found in standard works (3, 5,14,17).

4.2 C-bandingC-banding is the universal technique for demonstrating constitutive hetero-chromatin. In higher vertebrates this contributes to the complete characteriz-ation of the karyotype, and allows the study of certain types of chromosomeevolution. In those organisms (plants, invertebrates, and lower vertebrates)that lack euchromatic bands, C-banding is of fundamental importance for theidentification of chromosomes. Euchromatic bands cannot be demonstratedsatisfactorily in meiotic chromosomes, even in higher vertebrates, and hereagain C-banding is useful for chromosome identification.

Protocol 6. The BSG method for C-banding (20)

Equipment and reagents• 0.2 M hydrochloric acid (17.2 ml concern- • 2 x SSC at 60°C: 0.3 M sodium chloride,

trated acid/litre) 0.03 M trisodium citrate (17.53 g NaCI and. 5% barium hydroxide in distilled water at 8.82 g trisodium citrate per litre of distilled

50°C. Warm 40 ml distilled water to 50°C in water)—discard the heated working solu-a Coplin jar in a water-bath; a few minutes tion after an hour or twobefore use add 2 g Ba(OH)2.8H20 and stir • Giemsa: add 1 ml Gurr's Giemsa Improvedwell to dissolve. The layer of barium carbon- R66 (6DH) to 50 ml buffer pH 6.8, madeate that forms on the surface may be with Gurr's buffer tablets—discard after anskimmed off if necessary, but does not hour or two, as the dye precipitates afterusually cause any problems. Discard the dilutionsolution after an hour or so.

Method1. Allow the chromosome preparations to age for about a week before

C-banding.2. Put the chromosome preparations in 0.2 M hydrochloric acid for 1 h

at room temperature.

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3. Rinse briefly with distilled water.4. Place in the barium hydroxide solution for 1-5 min at 50°C.5. Rinse thoroughly with distilled water.6. Immerse the slides in 2 x SSC at 60°C for 1 h.7. Rinse with distilled water.8. Stain with Giemsa, 45 min.9. Rinse the Giemsa solution off with distilled water and carefully blot

the slides dry.10. Allow the slide to dry thoroughly for several minutes at room tempera-

ture, then mount in a synthetic neutral mountant (e.g. DPX).

It is characteristic of C-bands that they are heteromorphic, varying in sizebetween homologues in the same individual, and between different individu-als of the same species (14). Such variations may characterize inbred strains,as in mice (18), or distinct races of a species, as in certain grasshoppers (19).C-banded material appears to be selectively neutral in most species (and issometimes regarded as junk), and in humans, no convincing evidence hasbeen produced to associate heteromorphisms with clinical conditions such asinfertility, congenital abnormality, and mental retardation.

A C-banded mammalian metaphase spread is illustrated in Figure 1.

Figure 1. A C-banded human lymphocyte metaphase. Note the large blocks of hetero-chromatin on chromosomes 1, 9,16, and Y.

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4.3 G-bandingG-banding methods are the standard procedures for investigating the karyo-types of higher vertebrates, and are in routine use in clinical cytogeneticslaboratories throughout the world. The ASG method given here (21) is one ofthe standard methods for G-banding, and tends to give better chromosomemorphology than the somewhat more widely used trypsin methods.

Protocol 7. ASG method for G-banding (21)

Reagents« 2 x SSC at 60°C (see Protocol 6) • Giemsa (see Protocol SI

Method

1. Allow the chromosome preparations to age for about a week (accept-able results can probably be obtained between 3-14 days, or some-times much longer).

2. Incubate slides in 2 x SSC at 60°C for 1 h.

3. Rinse with distilled water.

4. Stain for 45 min.

5. Rinse thoroughly with distilled water.

6. Blot carefully, allow to dry thoroughly, and mount in a neutral moun-tant (e.g. DPX).

A human metaphase and karyotype banded by this procedure are illus-trated in Figure 2.

4.4 Ag-NOR staining for nucleolus organizing regionsThe sites of nucleolar organizers on chromosomes can be stained specificallywith silver (Ag-NOR staining). Ag-NOR staining is used to locate NORs onchromosomes of both animals and plants. Ag-NOR staining actually reflectsNOR activity at the preceding interphase, so that not all sites of genes forribosomal RNA are necessarily stained (14). Ag-NOR staining can be usedto study changes in ribosomal gene activity during embryonic development,during gametogenesis, and in cells with different genotypes and metabolicactivities.

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4; Microscopy of chromosomes

Figure 2. Human lymphocyte metaphase chromosomes (top) and karyotype (bottom),G-banded using the ASG technique. Reprinted with permission from Nature New Biol-ogy, 232, pp. 31-32, Copyright© 1971, Macmillan Magazines Ltd.

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Protocol 8. Ag-NOR staining (22)

Equipment and reagents• Hotplate at approx. 70°C • Colloidal developer: dissolve 2 g gelatin in. Silver nitrate: dissolve 4 g AgN03 in 8 ml 100 ml distilled water with gentle warming

distilled water—keep the solution in the and continuous stirring; when the gelatindark, and discard it if any blackening occurs has dissolved, add 1 ml pure formic acid

. Giemsa: 5% Gurr's Improved R66 (BDH) in (discard this soulution after about twobuffer pH 6.8, made with Gurr's buffer weeks)tablets (BDH)

Method

1. Slides are best used two to three days after the chromosomes havebeen spread.

2. Mix two drops of colloidal developer and four drops of silver nitratesolution in an Eppendorf tube. Pipette the mixture on to the chromo-some preparation, and cover with a coverslip.

3. Place the slide on a hotplate pre-heated to approx. 70°C. The slideshould be removed when the solution turns golden yellow, after 1-2min.

4. Remove the slide from the hotplate, and wash the coverslip off with astream of distilled water. Wash thoroughly with distilled water.

5. Counterstain with Giemsa for about 5 min.

6. Rinse with distilled water, blot, allow to dry thoroughly, and mountwith a neutral mountant (e.g. DPX).

Chromosomes showing Ag-NOR staining are illustrated in Figure 3.

4.5 CREST labelling of kinetochoresThis immunocytochemical method for labelling kinetochores differs from allthe others described above in that it cannot be used on standard alcohol: aceticacid fixed chromosome preparations, but requires cytocentrifuge preparationsthat have been made with minimal fixation (Protocol 4). CREST serum isobtained from patients with a particular form of the autoimmune disease sclero-derma; these vary considerably in the amount and type of anti-kinetochoreantibodies that they contain, and a new sample needs to be tested on materialthat is known to give a good reaction. Formerly it was necessary to obtainCREST sera from a friendly hospital rheumatology department, but nowadaysthey are also available commercially (often referred to as anti-centromereantibody, ACA). Because this is an immunocytochemical reaction involvingdifferent preparation procedures, it is difficult to combine CREST labellingwith routine banding to identify individual chromosomes, which can be aserious disadvantage of this procedure. If it is necessary to do kinetochore

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Figure 3. Ag-NOR staining of CHO metaphase chromosomes. The NORs are arrowed.Note the variability in size of the NORs. Counterstained with Giemsa.

labelling on alcohol:acetic acid fixed chromosomes, the Cd-banding procedureof Eiberg (23) could be tried, or alternatively, in situ hybridization withcentromeric DNA sequences, such as alphoid DNA in humans (24).

Protocol 9. CREST labelling of kinetochores

Equipment and reagents• Moist chamber: place paper towels on the

bottom of a plastic sandwich box, and soakthem with PBS, Add glass or plastic rods tokeep the slides above the wet towels; notethat the slides must be able to lie horizon-tally to avoid the reagents running away.

« CREST serum, obtainable from hospitalrheumatology departments, or commer-cially (The Binding Site)

. Pure methanol at -20"C

.PBS (see Protocol 4}. Bovine serum albumin (BSA)• Anti-human IgG, conjugated with FITC

(fIuorescein isothiocyanate)• Propidium iodide solution (approx. 1

mg/ml} or DAPI (2 mg/ml) in PBS• Antifadant mountant (e.g. Citifluor API,

Citifluor Ltd., or Vectashield, Vector Labora-tories I

Method1. Fix cytocentrifuge preparations of chromosomes for 10 min in methanol

at-20°C.2. Allow the slides to warm to room temperature and dry.3. Incubate the chromosome preparations for 30 min in a moist chamber

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A, T. Sumner and A. R. Leitch


at room temperature in CREST serum diluted with PBS containing 1%8SA. The optimal dilution for each serum must be found by experi-ence, but try 1:500 to start with. Put a drop of the diluted serum overthe area of the slides with the chromosomes and cover with a cover-slip (about 40 ul of the diluted serum for a 22 mm square coverslip).

4. Rinse the slides in three lots of PBS containing 1% BSA,5. Incubate for 30 min in the moist chamber in anti-human IgG, con-

jugated with FITC, diluted 1:5 with PBS containing 1% BSA.6. Rinse the slides in three lots of PBS containing 1% BSA.7. Counterstain with propidiurn iodide or DAPI.8. Rinse the slides with PBS.9. Mount with antifadant mountant, and seal the edges of the coverslip

with rubber solution.

A metaphase spread with the kinelochores labelled with CREST serum isshown in Figure 4.

5. In situ hybridizationDNA:DNA in situ hybridization, which involves hybridizing DNA probesdirectly to chromosomes (Figure 5), is an important method that has been putto many uses in genetic studies. These include:

(a) Mapping sequences to chromosomes, which is now a major tool ingenome mapping projects.

Figure 4. Imrrmnofluorescent labelling of kinetochores of CHO chromosomes withCREST serum, followed by FITC labelled anti-human IgG. The kinetochores appear aspairs of bright dots at the centromeres. Counterstained with ethidium bromide.

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Figure 5. In situ hybridization of a rye dispersed sequence AF1/4 (25) labelled with digoxigenin-11-dUTP to a root tip metaphase of aTriticum aestivum (wheat) x Secale cereale (rye) hybrid, Triticale cv TC400. (A) DAPI staining for DNA (blue fluorescence). The rye chromo-somes have bright DAPI positive subtelomeric heterochromatin, which become visible because of the denaturation step. Subtelomericheterochromatic bands are major C-bands in rye. (B) Sites of probe hybridization were detected using anti-digoxygenin-FITC (yellow/green fluorescence). Probe hybridization is restricted to the euchromatic regions of the rye chromosomes, (C) Double exposurefor DAPI and FITC fluorescence. Note that the rye chromosomes show label (cyan fluorescence) only on the euchromatic regions of thechromosome; the subtelomeric heteroctiromatin is unlabelled but fluoresces brightly with DAPI.


(b) Determining genome structure.(c) Understanding genome evolution and ancestry.(d) Analysing interphase nuclei.(e) Assessing sequence copy number.(f) Examining DNA condensation (11, 26-28).

The method enables sequences on more than one chromosome to bemapped at metaphase. The resolution of mapping is about 1 Mb, while onpachytene bivalents it is thought to be a few hundred kilobases. Less than 1 kbhas been detected in situ, and recently fibre spreading methods have enabledsequences separated by a few kilobases to be resolved (29). Furthermore sev-eral sequences can be detected simultaneously by using more than one probelabel. Using ratio or spectral imaging all human chromosomes can now besimultaneously painted in a single preparation (30).

5.1 Probe preparationThere are three main sources of DNA for use as probes for in situ hybrid-ization.

(a) Total genomic DNA. These probes have been used to perform genomicin situ hybridization (GISH) experiments to identify and localize alienchromosomes and chromosome sections introduced into crop plants (28)and to determine the ancestry of allopolyploid plants (27).

(b) PCR (polymerase chain reaction) generated probes. PCR probes havebeen used to make chromosome paints (31), to amplify specific DNAsequences (32), and to elongate sequences for in situ localization (33).

(c) Cloned sequences. These are still most commonly used and the vastmajority of sequences that are localized by in situ hybridization are firstcloned into plasmids, although cosmids, bacterial artificial chromosomes(Bacs), and yeast artificial chromosomes (Yacs) are increasingly used.The larger the cloned DNA fragment the more important it is to includeblocking DNA in the probe mixture. The blocking DNA allows in situsuppression of repeats in the probe and thus prevents in situ hybridizationsignal from labelled repeats occurring across the whole genome (34). Themethod is known as chromosomal in situ suppression.

The most commonly used DNA labelling methods are designed to incor-porate nucleotides conjugated to biotin, digoxigenin, or fluorochromesdirectly into the DNA. Sometimes, with elegant ratio labelling experiments,two different labels are incorporated into the same probe at known ratios. Byvarying the ratio between probes, and with electronic imaging methods(including spectral karyotyping), just two or three labels can uniquely identifymore than 20 probes simultaneously (30).

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Biotin is bound to nucleotides with a linker arm to minimize steric hin-drance of probe hybridization. The most commonly used biotin derivative isbiotin-11-dUTP, but other modified nucleotides are available. Biotin has astrong affinity to avidin which, when conjugated to a fluorochrome, is used todetect the probe hybridization sites. Digoxigenin is a natural steroid occurringin the plants Digitalis purpura and D. lanata. The derivative, digoxigenin-11-dUTP, is incorporated into DNA to make the probe. Antibodies againstdigoxigenin, typically conjugated to fluorochromes, are used to detect probehybridization sites.

Three fluorochromes conjugated directly to nucleotides are commerciallyavailable (Amersham), which are listed in order of decreasing sensitivity: fluor-escein (fluoresces yellow/green), rhodamine (fluoresces red), and coumarin(fluoresces blue). They are useful because after in situ hybridization and strin-gent washing the probe hybridization sites can be viewed directly and nodetection steps are necessary. Thus there are advantages of speed, they can beused easily in multiple labelling experiments, and there is little background.However, the sensitivity of the label is less than either biotin or digoxigenin(particularly the rhodamine and coumarin fluorochrome conjugates) and thishas restricted the use of these labels to the localization of highly reiteratedsequences. For this reason, and to explain the extra steps for biotin and digoxi-genin the protocols are restricted to these labels. However fluorochrome con-jugates are worth using where possible. Fluorochrome-11-dUTP conjugatesare incorporated into the probe in the same way as for digoxigenin-11-dUTP.

5.1.1 Probe labellingThere are many methods to incorporate labels into the DNA probe. The onedescribed here is nick translation (Protocol 10) which employs two enzymes,DNase I and E. coli DNA polymerase, that incorporate labelled nucleotidesalong both strands of the DNA probe by nicking the DNA and substitutingnucleotides. Commercially available enzyme mixtures have been optimized toproduce > 50% incorporation of label into DNA in about 90 minutes. In addi-tion they generate probe lengths of about 200-400 bp which is ideal for in situhybridization. Increasingly, polymerase chain reaction labelling of the probesis used, and during the amplification cycles, label is incorporated directly intothe probe by adding labelled nucleotides to the reaction mixture. If PCRgenerated probes are more than 1 kb long there may be probe penetrationproblems during the in situ reaction and signal strength reduced.

Protocol 10. Labelling of DNA by nick translation

Equipment and reagents• Water-bath at 15°C • Biotin labelled nucleotide: 0.4 mM biotin-• 100 mM dithiothreitol 11-dUTP (Sigma) made up from powder in. 0.3 M EDTA pH 8 100 mM Tris-HCI pH 7.5

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A. T, Sumner and A. R. Leitch

Protocol 10. Continued• DNA for labelling: optimal concentration of

DNA for labelling is 0.5-1 ug/ml; ideal DNAlength is greater than 500 bp

. 10 x nick translation buffer: 0.5 M Tris-HCIpH 7.8, 0.05 M MgCI2, 0.5 mg/ml (w/v)bovine serum albumin, nuclease-free

• Unlabelled nucleotide mixture: make 0.5mM solutions of each nucleotide (dCTP,dGTP, and dATP; Sigma) in 100 mMTris-HCI pH 7.5 and prepare a 1:1:1 mixture

. 1 x TE: dilute 100 x TE (1 M Tris-HCI pH 8,0.1 M EDTA) with water

. DNA polymerase l/DNase I: 0.4 U/u1 (GibcoBRL)—do not leave at room temperature; itis active at 15°C and is damaged at highertemperatures

. Digoxigenin labelled nucleotide mixture:mix digoxigenin-11-dUTP (1 mM stocksolution; Boehringer Mannheim) and dTTP(1 mM stock) to a final concentration of 0.35mM digoxigenin-11-dUTP and 0.65 mMdTTP

> 3 M sodium acetate. 70% and 100% ethanol (ice-cold)

Method

1. In a 1.5 ml Eppendorf tube prepare the nick translation solution asfollows: 5 ul 10 x nick translation buffer, 5 ul unlabelled nucleotidemixture, either 1 ul digoxigenin labelled nucleotide mixture, or 2.5 ulbiotin labelled nucleotide, 1 ul 100 mM dithiothreitol, a quantity ofsolution containing 1 ug DNA, water to make up the total volume to45 ul.

2. Add 5 (ul DNA polymerase l/DNase I solution, mix gently, and cen-trifuge briefly.

3. Incubate at 15°C for 90 min.

4. Stop the reaction by adding 5 ul 0.3 M EDTA pH 8.

5. Precipitate the DNA by adding 5 ul 3 M sodium acetate (or 5 ul 4 MLiCI) and 150 ul ice-cold ethanol. Place the DNA in the freezer (-20°C)overnight or on dry ice for 1-2 h.

6. Centrifuge the tubes at-10°C for 30 min at 12000 g.

7. Discard the supernatant by inverting the Eppendorf tube on to freshtissue paper in a single action.

8. Wash the pellet by adding 0.5 ml ice-cold 70% ethanol and then spinfor 5 min. Tip off solution as in step 7.

9. Leave the pellet to dry (several hours at room temperature, less undervacuum).

10. Resuspend the DNA in 1 X TE (typically 10 ul to give an estimatedconcentration of 0.1

5.1.2 Checking label incorporationAfter DNA labelling it is advisable to check label incorporation either byusing a dot blot (see Protocol 11) or by running a gel and observing retarda-tion of mobility in labelled samples. The latter is particularly useful if directlabel fluorochromes are used in PCR labelling reactions.

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Protocol 11. Checking incorporation of the probe

Equipment and reagents• Hybond N* (Amersham) • Detection of biotin incorporation: 1:500• Autoclave bags dilution of alkaline phosphatase-avidin D. Buffer 1: 0.1 M Tris-HCI pH 7.5, 0.15 M NaCI (Vector Laboratories) in buffer 1.Buffer 2: 0.5% (w/v) blocking reagent • Detection of digoxigenin: 1:5000 dilution of

(Boehringer Mannheim) in buffer 1, dis- anti-digoxigemn conjugated to alkalinesolved by heating the solution to 50-70°C phosphatase (Boehringer Mannheim) infor at least 1 h—the solution can be stored buffer 1at 4°C for up to one month • NBT (4-nitroblue tetrazolium chloride)/BCIP

. Buffer 3: 0.1 M Tris-HCI pH 9.5, 0.1 M NaCI, <5-bromo-4-chloro-2-indolyl-phosphate)0.05 M MgCI2 stable mixture (Gibco BRL)

Method

1. Cut the Hybond N+ membrane to the size required.

2. Soak the membrane in buffer 1 for 5 min and then blot dry betweenfilter paper.

3. Load the DNA on to the membrane (1 ul) and leave to dry for 5 min.

4. Place the membrane in buffer 1 for 1 min and then into buffer 2 for 30min. Shake gently during this period.

5. Drain the membrane and place in a Petri dish.

6. Add appropriate alkaline phosphatase conjugate in buffer 1 to themembrane, cover with plastic cut from an autoclave bag, and incubateat 37 °C for 30 min.

7. Wash the membrane in buffer 1 three times for 5 min.

8. Transfer the membrane to buffer 3 for 2 min.

9. Add NBT/BCIP mixture, apply fresh plastic cover, and incubate in thedark for 10 min for the colour to develop fully. Wash the membrane tostop the reaction in water and air dry.

10. Select only those probes that produce a good clear dot.

11. Code the probes with a strong signal so that it is easy to reference theprobe to the dot blot. Discard weakly labelled probes.

5.2 In situ hybridization reactionThe in situ hybridization reaction is carried out over a two day period. Thereaction has a large number of steps.

(a) RNase treatment. This pre-treatment removes cellular RNA so that theprobe can only bind to cellular DNA, most of which is chromosomal.

(b) Pepsin treatment. This pre-treatment is optional and should be omitted ifpossible. It increases the sensitivity of in situ hybridization, and is useful,

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sometimes essential, in detecting low copy sequences. However, chromo-somes become more vulnerable to damage during denaturation steps andthe treatment may add unnecessary steps to the protocol.

(c) Denaturation. Denaturation of the chromosomal DNA is a critical stepwhich should be monitored carefully. The denaturation solution containsformamide which destabilizes DNA and reduces the melting temperatureof the DNA by about 0.6 X the percentage of formamide in the solution.In contrast, the concentration of cations (Na+) increases the melting tem-perature by about 17 X log molarity of cations. Thus a balance of salts,formamide, and temperature regulate the denaturation conditions (formore information see ref. 35). Too much denaturation and the chromo-somes are stripped of DNA; too little and the DNA is not denatured. Theprotocol describes general conditions. For new material try denaturingchromosomes at a range of temperatures, wash slides, dehydrate themthrough an ethanol series, and examine the condition of the chromosomeswith phase or preferably Nomarski phase-contrast microscopy or follow-ing staining with DAPI. Choose the hottest solution that maintainschromosome structure.

(d) Hybridization and hybridization mixture. The hybridization mixture con-tains probe DNA, blocking DNA, salts, formamide, dextran sulfate, andsodium lauryl sulfate (SDS). The dextran sulfate increases the hybridiza-tion reaction rate by a factor of three and works by forming a matrix inthe hybridization mixture which concentrates the probe without affectingthe denaturation conditions. The probe(s) are typically labelled withfluorochromes, digoxigenin, biotin, or ratios of each. The blocking DNAhas different roles in different experiments and is typically used at con-centrations of 2-250 times the probe concentration. In all experiments itreduces non-specific probe hybridization. With CISS and chromosomepainting experiments, the blocking DNA comes from the organism understudy and the blocking DNA increases the specificity of probe hybridiza-tion. With GISH experiments, the blocking DNA is obtained from one ofthe parents in the hybrid organism being studied and once again increasesthe specificity of probe hybridization (31, 34). Probe and blocking DNAneed to be denatured in the hybridization mix by heating, and applied tothe material for in situ hybridization overnight at 37 °C.

(e) Stringent washing. This wash is designed to strip away weakly bound andnon-specifically bound probe which may occur during the overnight insitu hybridization reaction. The solution contains salts and formamide atconcentrations that destabilize weakly bound probes leaving only stronglybound probe hybridized. The protocol leaves a stable sequence identity ofgreater than 80-85%. Increasing the temperature of this wash can furtherreduce background and non-specific labelling but the strength of desirablesignal may also decline.

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(f) Detection. In double label experiments using biotin and digoxigenin thebiotin is detected with avidin conjugated to one fluorochrome (here Cy3)while digoxigenin is detected with an antibody conjugated to anotherfluorochrome that fluoresces at a different wavelength (here FITC). Thechoice of fluorochromes arises because Cy3 is only obtained conjugatedto avidin and it is a particularly good fluorochrome.

(g) Counterstain. Use only DAPI fluorescence for a chromosomal counter-stain unless there are compelling reasons to use propidium iodide. Pro-pidium iodide results in a great loss of information due to fluorescenceacross a wide spectrum.

(h) Antifadant. This increases the stability of the fluorochromes, e.g. FITC,Cy3, and DAPI which is essential for photography.

Protocol 12. In situ hybridization

Equipment and reagents• Humid chamber. Place 2 x SSC soaked tis-

sues in a floating box with a lid. Put into thebox two glass rods on to which the slideswill be placed; the rods will keep the slidesabove the wet tissues. Float the box in awater-bath at 37°C (or place in a 37°Croom) and allow the box to equilibrate atthe temperature.

• Plastic coverslips: made from plastic auto-clave bags cut to an appropriate size tocover the material, but not to overlap theedge of the slide

. Water-baths at 37°C and 70°C• 20 x SSC pH 7: 3 M NaCI, 0.3 M sodium

citrate. 2 x SSC: dilute 20 x SSC with water (1:9)• RNase A: prepare a stock solution by dis-

solving 10 mg/ml of DNase-free RNase in10 mM Tris-HCI pH 7.5, 15 mM NaCI. Boilfor 15 min and allow to cool. Store frozen inaliquots. For use dilute to 100 ug/ml RNaseA in 2 x SSC.

• 0.01 M HCI, optional step• 1 ug/ml pepsin in 0.01 M HCI: 3200-4500

U/mg protein (porcine stomach mucosa)(Sigma)

• Pre-hybridization fix: ethanol, glacial aceticacid (3:1), freshly prepared

. Ethanol series: 70%, 90%, 100% ethanol• Ice-cold ethanol series: 70%, 90%, 100%

ethanol stored on ice• Formamide denaturation solution: heat

70% (v/v) formamide in 2 x SSC (70 mlformamide, 10 ml 20 x SSC, 20 ml water)to exactly 65°C (solution temperature) ina water-bath—prepare immediately before

• Formamide: deionized, high grade for-mamide is used, stored frozen at -20°C (ifall the formamide does not freeze, buyfresh formamide)

• 50% (w/v) dextran sulfate (M, 500000): dis-solve in water at 37°C overnight and storein aliquots at -20°C—warm to 37°C toreduce viscosity before use

• Blocking DNA: store at -20°C, defrost andvortex well before use. (a) For mappingshort cloned sequences: salmon totalgenomic DNA (autoclaved, 10 min). (b) ForGISH experiments: total genomic DNA(autoclaved, 10 min) from the organismunder study, (c) For chromosome paintingand CISS experiments: total genomic DNA(autoclaved, 10 min) from the organismunder study.

• Probe DNA-dig: digoxigenin labelled DNA(0.1 ug/ml) stored in freezer, defrost andvortex well before use

• Probe DNA-bio: biotin labelled DNA (0.1ug/ml) stored in freezer, defrost and vortexwell before use

• 10% (w/v) SDS in water• Hybridization mix (see Table 1)• Stringent wash: 20% formamide in 0.1 x

SSC (20 ml formamide, 0.5 ml 20 x SSC,79.5 ml water)

• 4 x SSC/Tween: dilute 4 parts 20 x SSCwith 16 parts water and add 0.2% (v/v)Tween 20

• Bovine serum albumin (BSA). BSA block: 5% (w/v) BSA in 4 x SSC/Tween

20• Detection reagent: prepare a mixture of 10

(ug/ml anti-digoxigenin-FITC and 5avidin-Cy3 in BSA block

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Protocol 12. Continued• DAPI: stock solution (100 (ig/ml DAPI in

water), stored in aliquots at -20°C—dilutestock solution to 2 ug/ml DAPI in 2 x SSC

• Anti-digoxigenin-FITC (Boehringer Mannheim)(digoxigenin detection reagent): make stocksolutions of 500 ug/ml in sterile distilledwater according to manufacturer's instruc-tions, and store aliquoted in freezer (freezesolution once only)

. Cy3-avidin (Amersham) (biotin detectionreagent): supplied at stock concentration (1mg/ml)—store aliquoted in freezer (freezesolution once only)

. Antifadant: Vectashield antifadant reducesbleaching of fluorochromes; also availablewith DAPI added (Vector Laboratories)

A. Day 7: slide pre-treatments and hybridization steps

1. Place slides in an oven at 37°C overnight.

2. Add 200 ul RNase A, cover with a plastic coverslip, remove airbubbles, and incubate for 1 h at 37°C in a humid chamber.

3. Wash slides in 2 x SSC, three times for 5 min each.

4. If pepsin is used, place material in 0.01 M HCI for 2 min.

5. Optional step. Add 200 ul pepsin solution, cover with a plastic cover-slip, and incubate for 10 min at 37°C.

6. If pepsin is used, stop reaction by placing in water for 2 min, andwash in 2 x SSC, twice for 5 min each.

7. Place material in pre-hybridization fix for 10 min.

8. Dehydrate for 3 min each in 70%, 90%, and 100% ethanol, and thenair dry.

9. Prepare the hybridization mix as shown in Table 1 prior to denaturingslides.

10. Denature the hybridization mix at 70°C for 10 min, vortex briefly, andplace on ice.

11. Denature slides. Place slides in formamide denaturation solution at65°C for 3 min. The temperature needs to be determined empiricallyfor your material (see text). This is one of the most crucial steps.When using a standard Coplin jar, each slide causes a reduction inthe temperature of the formamide solution of about 2°C. Thereforedenature slides one at a time and note the solution temperaturechange.

12. Place slides in ice-cold ethanol series, 3 min in each, and air dry.

13. Add 40 ul of the freshly denatured hybridization solution and coverwith a plastic coverslip. Ensure there are no air bubbles.

14. Incubate overnight at 37°C in the humid chamber.

B. Day 2: post-hybridization washing and label detection

1. Float coverslips off in 2 x SSC at 35-42°C.

2. Place slides in stringent wash, twice for 5 min each, at 42°C.

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3. Wash slides in 2 x SSC at 42°C, three times for 3 min each.

4. Take Coplin jar out of water-bath and leave to cool for 5 min.

5. Wash slides in 2 x SSC three times for 3 min each.

6. Place slides in 4 x SSC/Tween for 5 min.

7. Drain but do not allow slide to dry. Add 200 ul BSA block to each slideand apply a plastic coverslip, incubate for 5 min.

8. Remove coverslip and drain but do not allow slide to dry. Add 30 uldetection reagent per slide. Replace coverslip and incubate for 1 h at37°C in a humid chamber.

9. Wash slides in 4 x SSC/Tween at 37°C, three times for 8 min each.

10. Drain solution, but do not allow slide to dry. Add 100 ul DAPI per slideto counterstain the chromosomes, cover with plastic coverslip, andincubate for 10 min.

11. Rinse briefly in 4 x SSC/Tween.

12. Drain but do not allow slide to dry. Apply antifadant solution.

13. Place a thin coverslip (preferably UV transparent and of high quality)over material. Gently squeeze excess antifadant from the slide withfilter paper.

Table 1. Composition of hybridization mixture

Solution

Formamide50% (w/v) dextran sulfate20 x SSCProbe DNA-dig (0.1 p-g/pl)Probe DNA-bio (0.1 ug/Ml)Blocking DNA (1 ug/ul)10%(w/v)SDSWater

To prepare 40 ul

20 Ml8 ul4Ml1 ul(100 ng/slide)1 ul(100 ng/slide2 Ml (2 ug/slide)0.5 Ml3.5 ul

Final concentration

50%10%2 X2.5ng/ul2.5 ng/ul250 ng/ul1.25%

Use 40 (ul per slide. Adjust ratios appropriately for different volumes.

6. Observation and recording of images ofchromosomes

Many of the features that can be demonstrated on chromosomes are at ornear the limit of resolution of light microscopy. A good quality microscope,equipped with high quality lenses and properly adjusted, is therefore neces-sary for observing chromosomes. A high quality camera is also required,and increasingly, confocal microscopes and CCD cameras are being used; the

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latter are particularly useful for fluorescence studies, particularly in in situhybridization, as they can be used to obtain multicoloured images at highresolution and high sensitivity (as well as high price!).

Practical details of how to set up a microscope to obtain optimal resultshave been given elsewhere (36) (also Chapter 1). Requirements for observa-tion of chromosomes stained with absorbing dyes, and those stained withfluorochromes are, in many ways, rather different. However, one importantfactor for the study of chromosomes is the use of flat field objectives. Theseare not only necessary for photography and other image recording systems,but are also valuable for direct observations. It is excessively tiresome if thefocus has to adjusted continually to observe chromosomes in different parts ofthe field and comparison between different chromosomes becomes muchmore difficult if only one is in-focus at a time.

6.1 Observation of banding with absorbing dyesA low power (x 10) objective is necessary to locate metaphase spreads onthe slide, where they may be quite rare compared with the interphase nuclei,but this magnification is totally inadequate to see banding. In fact, a X 90or X 100 oil immersion objective is necessary to assess the quality of the pre-paration, and to make definitive observations. The contrast of bandedchromosomes can be improved by optical means, either by the use of phase-contrast or differential interference (Nomarski) microscopy, or by the use offilters. Phase-contrast has been used for improving the visibility of Giemsastained R-bands, which are sometimes rather pale, and can also be used withAg-NOR staining if the chromosomes have not been counterstained. In thelatter case, the Ag-NORs appear as bright objects against a darker back-ground. Green filters can be used to increase the visibility and contrast ofGiemsa stained chromosomes.

6.2 Observation of fluorescent chromosomesFluorescently stained chromosomes generally have a low level of fluorescence,and therefore a fluorescence microscope of the highest quality is needed.The characteristics of such a system are described in detail elsewhere (37)(Chapter 6). The important features are the greatest possible efficiency ofillumination (ideally a 100 W mercury lamp), and of collection of the emittedfluorescence. These are achieved by using epi-illumination (incident illumina-tion through the objectives), and objectives of the highest possible light trans-mission, i.e. high numerical aperture, flat field, fluorescence objectives. Theseare available from the major microscope manufacturers especially for fluor-escence work. It is worth purchasing oil immersion objectives of both approx-imately X 50 and X 100 magnification, as fluorescence microscopy requirescontinual changing between these two magnifications, the lower being usedfor detailed scanning of the slide, and the higher for observation.

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Fluorescence microscopy requires the use of exciter filters, to ensure thatonly light of the required wavelengths reaches the specimen to excite fluor-escence, and barrier filters, to cut out the exciting light and to transmit onlythe fluorescence. With epi-illumination, these filters are combined in a singlemodule with a dichroic reflector which enables the epifluorescence system towork by reflecting short wavelengths and transmitting long wavelengths. It isvital to use the correct filter combinations for the fluorescence to be studied(Table 2). Unfortunately, there is no universal system for designating filtermodules for fluorescence, and each manufacturer has its own system. It isalways advisable to try out any objectives and filter modules on the types ofspecimen you are interested in, to see which gives the best results, beforespending any money.

A serious problem in all fluorescence microscopy is fading of fluorescenceduring illumination. Special mountants that retard the fading of fluorescenceare now available from various sources (Citifluor; Vector), and the use of

Table 2. Useful fluorochromes for use in cytogenetics"

Excitation(max, nm)

Fluorochromes that stains DNA

Propidium iodide 340, 530

Emission Fluorescence(max, nm) colour

DAPI 355

Chromomycin A3 430

615

450

570

Red

Blue

Yellow

Fluorochromes conjugated to probe detection systems

Fluorescein 495 515 Green(FITC)

Cy3

Coumarin(AMCA)

550

350

570

450

Orange

Blue

Microscopefittersb

BP 525-560CBS 580LP590BP 340-380CBS 420LP420BP436CBS 455LP470

BP 450-490CBS 510LP520BP515-560C

CBS 580LP590BP 340-380CBS 420LP420

aThe microscope filters are for visualizing single fluorochromes. Double and triple bandpass filtersenable combinations of fluorochromes to be visualized simultaneously and can be obtained fromleading microscope suppliers.bBP = bandpass filter, light above or between wavelength given is transmitted. CBS = chromaticbeam splitter, light above wavelength given is transmitted, below it is reflected. LP = long pass filter,light above wavelength given is transmitted.cUse rhodamine filter block.

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some of these has been described in some of the protocols given above. Twopoints should be made about such mountants. First, although they do retardthe rate of fading (but do not usually stop it entirely), they may also reducethe brightness of the fluorescence, which may limit their value. Secondly, thereis no universal antifadant, so that what works well with one fluorochrome maybe useless with another. Some fluorochromes, such as quinacrine, cannot bestabilized by any antifadant; on the other hand, it has been found that storageovernight or for a few days before observation greatly stabilizes the fluor-escence, as with DAPI and chromomycin.

6.3 Photography of chromosomesPhotography of chromosomes provides a permanent record, not only for pub-lication and to provide a reference in clinical studies, but also for detailedanalysis of banding and in situ hybridization patterns. General principles forphotographing banded chromosomes have been described by Davidson (38).The first requirement, of course, is that the microscope should be properly setup (36) (Chapter 1). It is necessary to take a test strip both when using newequipment and when trying out a new type of film or developer. Exposuresshould be varied on either side of those recommended by the manufacturer ofthe film, or prescribed by the automatic exposure meter. The effect of variousfilters should also be tested. All relevant data must be recorded, so that theoptimal exposure can be used in the future.

6.3.1 Black and white photographyFor photographing chromosomes stained with absorbing dyes, a slow blackand white film such as Kodak Technical Pan or Ilford Pan F is quite adequate,as there is no problem with the staining being bleached, and these slow filmsproduce the finest grain. For photographing fluorescence, the fastest possiblefilm is desirable, because of the problem of fading, and the low light levelsinvolved. Formerly it was impossible to obtain a reasonable film speed (400ASA or more) without excessively coarse grain, but nowadays such films notonly have a reasonably fine grain, but can also be rated at a higher speed usingsuitable development—consult the manufacturers' instructions on this point.For fluorescence work it is essential to have a light meter that can select aspecific area of the specimen, i.e. part of a fluorescent chromosome, as it isimpossible to calculate the correct exposure from an area consisting largely ofblack background. A particular problem with photographing fluorescence isreciprocity failure: in effect, the longer the exposure, the slower the effectivefilm speed. For photographing very dim fluorescence, therefore, it may benecessary to rate the film at half, or even a quarter, of its usual speed.

6.3.2 Colour photographyColour photography can be very valuable for fluorescence work, particularlywhere two or more different colours have to be distinguished, as in double

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labelling in situ hybridization. Here again, films have been greatly improvedin recent years, and it is possible to get high speed colour films withoutexcessively coarse grain.

The recording of images by colour photography offers many advantagesover black and white photography:

(a) Multiple fluorochrome labels can be distinguished.

(b) Dark-room time is eliminated.(c) Film processing can be done quickly.

For epifluorescence microscopy it is necessary to have a high speed, finegrain film with high colour contrast. The films we use are Fujicolor 400 andFujichrome 400. Slide films (Fujichrome 400) give the most accurate represen-tation of colour, but this may not be as important as clear colour distinctionbetween fluorochromes. They also have smaller grain sizes than print filmsand can be processed anywhere, even in the laboratory. The main disadvan-tage is that slides are difficult to analyse.

Print films (Fujicolor 400) are convenient, as several copies of prints can bemade as a matter of routine and prints are easily analysed. In addition, whenusing print films there is more flexibility with exposure times because simpleadjustments during printing can generate acceptable images from negativeswith widely different densities. However, printing can be a problem as a photo-grapher needs to be found who has the facility to print the exposure times andfilter settings on the back of each print. Most towns have such photographers.In consultation with the photographer the optimal settings and ranges areestablished for printing. In a short time it is then only necessary to drop offthe films and pick up the prints. But setting up the necessary dialogue with thephotographer takes time.

6.4 Other methods of image captureIncreasingly, electronic images are being captured to record images, in par-ticular those from fluorescent in situ hybridization experiments. This is due toan increasing use of confocal microscopes, low light cameras, and imageenhancement. In addition, images recorded on photographic film are beingscanned into an electronic image for printing with high contrast on colourprinters.

6.4.1 Confocal microscopesThe confocal microscope offers advantages in that it can be used to obtainhigh resolution images, and if a sufficiently small aperture is used, much of theout-of-focus information can be removed. The microscope scans the specimenwith a spot of laser light at an appropriate wavelength to excite fluoro-chromes. Fluorescent light passing through the confocal aperture is recordedwith a photomultiplier and displayed point by point on to an appropriate high

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resolution monitor. Confocal microscopy is used routinely by many laborato-ries and the technique offers certain advantages.

(a) The brief period of fluorochrome excitation required for producing a con-focal image (usually about 2 sec) reduces the bleaching of fluorochromes.

(b) The digital images recorded by the confocal microscope can be stored andrecombined enabling easy alignment of different images.

(c) Instant images are obtained, whereas conventional microscopy requiresfilm processing, which can take days.

(d) The resolution of images may, if the instrument is used at its highest pos-sible performance, be higher.

(e) Optical sections can be taken and three-dimensional objects reconstructed.

The potential increased resolution and the possibility of reconstructionfrom optical sections does mean that in some circumstances the confocal isextremely powerful. However there are several major disadvantages.

(a) In practice a confocal microscope slows down analysis of slides and fewercells will be examined.

(b) The images recorded can be subjected to too much manipulation whichcan distort or lose information.

(c) The microscopes are very expensive to buy and run.

For further information see Chapter 6, Section 7.3.

6.4.2 Low light (CCD) camerasThese cameras record fluorescent images electronically (see Chapter 6, Sec-tion 7.4). They have one major advantage over conventional cameras: theacquisition of instant images. However they have all the disadvantages of aconfocal microscope above (except running costs). In theory, fluorescence notvisible to the eye can be recorded and imaged, although in our experience,this ability is of little use. Background and non-specific signals tend to beimaged as well as the real signal, so that the latter is hard to distinguish. Thusimaging is restricted to material where the signal is visible. An instant imagecan be obtained and processed, but there are the dangers of image distortionor misinterpretation. If obtaining an instant image is the desired feature, thenbe careful to purchase a suitable camera. Do not compare an electronic imagewith what can be seen down the eyepiece of the microscope, because signalbrightness and image size make this difficult. Compare a print of an imagetaken from a low light camera with a print from a conventional camera. Checkresolution, pixel size, chromosome, and signal clarity. Do not take too muchnotice of the brightness of colours, which can, if required, be changed on aprint either photographically or electronically. Be careful to ensure that instantimages are really needed, and then be careful which camera is purchased.

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6.4.3 Image processingAll electronic images can be processed and printed at high quality. In addition,images obtained by conventional microscopy can be stored electronically,processed, and imaged. Equipment for doing this is now widely available. Inall cases published electronic images are brilliant in intensity and colour, pre-cise, and usually impressive. However, image processing is one of the mostdangerous tools available to cytogeneticists. It requires scrupulous adherenceto ethical manipulation approaches as almost anything can be 'created' or'destroyed' using modern software. We would recommend that the onlyacceptable manipulation is that which affects the whole image uniformly.

References1. Schwarzacher, T. S. and Leitch, A. R. (1994). In Methods in molecular biology (ed.

P. Isaac), Vol. 28, p. 153. Humana Press, Totowa, NJ.2. Macgregor, H. C. and Varley, J. M. (1988). Working with animal chromosomes

(2nd edn). John Wiley & Sons, Chichester.3. Barch, M. J. (ed.) (1991). The ACT cytogenetics laboratory manual (2nd edn).

Raven Press, New York.4. Bennett, M. D. and Smith, J. B. (1976). Phil. Trans. R. Soc. Land. B, 274, 227.5. Rooney, D. E. and Czepulkowski, B. H. (ed.) (1992). Human cytogenetics: a prac-

tical approach (2nd edn). Oxford University Press, Oxford.6. Gosden, J. R. (ed.) (1994). Chromosome analysis protocols. Humana Press,

Totowa, NJ.7. Chandley, A. C., Speed, R. M., and Ma Kun. (1994). In Chromosome analysis pro-

tocols (ed. J. R. Gosden), p. 27. Humana Press, Totowa, NJ.8. Jeppesen, P. (1994). In Chromosome analysis protocols (ed. J. R. Gosden), p. 253.

Humana Press, Totowa, NJ.9. Stenman, S., Rosenqvist, M., and Ringertz, N. R. (1975). Exp. Cell Res., 90, 87.

10. Pan, W. H., Houben, A., and Schlegel, R. (1993). Genome, 36, 387.11. Leitch, I. J. and Heslop-Harrison, J. S. (1993). Genome, 36, 517.12. Spurbeck, J. L., Zinsmeister, A. R., Meyer, K. J., and Jalal, S. M. (1996). Am. J.

Med. Genet., 61, 387.13. Worton, R. G. and Duff, C. (1979). In Methods in enzymology (ed. W. B. Jakoby

and I. H. Pastan), Vol. 58, p. 322. Academic Press, New York.14. Sumner, A. T. (1990). Chromosome banding. Unwin Hyman, London.15. Bickmore, W. and Sumner, A. T. (1989). Trends Genet., 5, 144.16. Holmquist, G. P. (1989). J. Mol. Evol., 28, 469.17. Schweizer, D. and Ambros, P. F. (1994). In Chromosome analysis protocols (ed.

J. R. Gosden), p. 97. Humana Press, Totowa, NJ.18. Davisson, M. T. (1989). In Genetic variation and strains of the laboratory mouse

(ed. M. F. Lyon and A. G. Searle), p. 617. Oxford University Press, Oxford.19. Webb, G. C., White, M. J. D., Contreras, N., and Cheney, J. (1978). Chromosoma,

67, 309.20. Sumner, A. T. (1972). Exp. Cell Res., 75, 304.21. Sumner, A. T., Evans, H. J., and Buckland, R. A. (1971). Nature New Biol, 232, 31.

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22. Howell, W. M. and Black, D. A. (1980). Experientia, 36, 1014.23. Eiberg, H. (1974). Nature, 248, 55.24. Mitchell, A., Jeppesen, P., Hanratty, D., and Gosden, J. (1992). Chromosoma, 101,

333.25. Francis, H. A., Leitch, A. R., and Koebner, R. M. D. (1995). Theor. Appl. Genet.,

90, 636.26. Ferguson-Smith, M. A. (1991). Am. J. Hum. Genet., 48, 179.27. Leitch, I. J., Parokonny, A. S., and Bennett, M. D. (1996). In Chromosomes today

(ed. N. Henriques-Gil, J. S. Parker, and M. J. Puertas), Vol. 12, p. 333. Chapmanand Hall, London.

28. Schwarzacher, T., Anamthawat-Jonsson, K., Harrison, G. E., Islam, A. K. M. R.,Jia, J. Z., King, I. P., et al. (1992). Theor. Appl. Genet., 84, 778.

29. Zhong, X. B., Fransz, P. F., Wennekes-Vaneden, J., Zabel, P., Vankammen, A.,and De Jong, J. H. (1996). Plant Mol Biol. Rep., 14, 232.

30. Schrock, E., Dumanoir, S., Veldman, T., Schoell, B., Wienberg, J., Ferguson-Smith, M. A., et al. (1996). Science, 273, 494.

31. Telenius, H., Pelmear, A. H., Tunnacliffe, A., Carter, N. P., Behmel, A., Fergu-son-Smith, M. A., et al. (1992). Genes Chromosomes Cancer, 4, 257.

32. Pearce, S. R., Harrison, G., Li Dongtao, Heslop-Harrison, J. S., Kumar, A., andFlavell, A. J. (1996). Mol. Gen, Genet, 250, 305.

33. Cox, A. V., Bennett, S. T., Parokonny, A. S., Kenton, A., Callimassia, M. A., andBennett, M. D. (1993). Ann. Bot., 72, 239.

34. Lichter, P., Tang, C. C, Call, K., Hermanson, G., Evans, G. A., Housman, D., etal. (1990). Science, 247, 64.

35. Meinkoth, J. and Wahl, G. (1984). Anal. Biochem., 138, 267.36. Bradbury, S. (1989). Introduction to the optical microscope. Oxford University

Press, Oxford.37. Ploem, J. S. and Tanke, H. J. (1987). Introduction to fluorescence microscopy.

Oxford University Press, Oxford.38. Davidson, N. R. (1973). /. Med. Genet, 10,122.

184

ImmunohistochemistryMICHAEL G. ORMEROD and SUSANNE F. IMRIE

1. IntroductionImmunohistochemistry utilizes antibodies to localize specific products in tissuesections. Briefly, a tissue section is incubated with a labelled antibody, thesection is washed, and the site of reaction of the antibody is identified byvisualizing the label.

A variety of histochemical techniques have been used for many years toidentify certain constituents of cells. These methods lack the specificityobtained from an antibody and it is this property which enables a worker tomap precisely the distribution of a particular product in a tissue. Aside fromits power as a tool in research, immunohistochemistry has found an increasingrole in diagnostic histopathology with particular application to the diagnosisof tumours. The range of applications of the method has been advancedimmeasurably by the development of technology for the production of mono-clonal antibodies. This has produced a wide range of antibodies, many ofwhich have specificities which would have been difficult, if not impossible, toobtain from conventional antisera.

The first part of the chapter (Sections 2-5) give the background to the tech-niques. The later sections describe the procedures in detail. Section 2 brieflydefines an antibody and its structure, and describes the difference betweenpolyclonal sera and monoclonal antibodies. The possible effects of tissue pro-cessing on the antigen are considered in Section 3. A wide variety of differentlabels may be employed; these are described and compared in Section 4.Section 5 describes and compares in general terms the different methods usedfor applying antibodies to tissue sections.

Section 6 contains the details of the experimental methods used withenzyme labelled antibodies. The use of controls and problem solving are dis-cussed in Section 7 and ways of visualizing two antigens simultaneously inSection 8.

Although the chapter concentrates on the use of antibodies, a similar tech-nique can be applied using any reagent that shows a similar specificity. Forexample, lectins can be used to identify certain groups of carbohydrates.

Most of the chapter describes techniques for staining sections cut from

5

Michael G. Ormerod and Susanne F. Imrie

tissue. The same methods can also be applied to cytological preparations anda short section (Section 9) on staining smears is included.

Section 10 contains a brief discussion on quantification and Section 11describes a staining tray—the only special piece of equipment needed.

For further reading, several books on immunohistochemistry have beenpublished. Most of these give details of the application of the technique toparticular problems (1-5).All the reagents listed in this chapter can be purchased from Sigma Ltd.unless otherwise stated.

2. AntibodiesAntibodies, collectively called immunoglobulins (Igs), comprise approxi-mately 20% of the proteins in human plasma. They are produced by plasmacells and can exist in millions of different forms. During an immune response,the foreign body (antigen) stimulates division of those plasma cells responsiblefor producing an Ig reactive with that particular antigen. This enables theanimal to produce large numbers of specific antibodies. Any animal in anormal environment continually undergoes immune responses and its plasmawill contain antibodies directed against thousands of different antigens.

2.1 Immunoglobulin structureThe basic structure of an Ig is a dimer, each half containing two polypeptidechains, one called heavy (containing ~ 440 amino acids), the other light(containing ~ 220 amino acids). The chains are linked by disulfide bonds(Figure 1). The heavy chain has an invariant (constant) region, which deter-mines the class of the Ig, and a variable region. The latter, in conjunction withthe variable region on the light chain, forms the site that binds the antigen.Light chains can be of two classes, kappa or lambda.

The major classes of Igs are listed in Table 1. The IgG class is further sub-

Figure 1. A simplified representation of an Ig molecule showing the antigen binding siteand the sites of cleavage of papain and pepsin. — represents a disulfide bond.

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5: Immunohistochemistry

Table 1. The major classes of immunoglobulins

Name Total Ig in blood (%) Comment

IgGIgAIgMigDlgE

80155< 1<1

Major antibody in immune sera.Found in sero-mucous secretions.Produced early in immune response, pentameric.Present on lymphocyte surfaces.Responsible for allergic reactions.

divided (IgG1, IgG2, etc.). During an immune response, the bulk of the Igs inthe plasma are IgGs. Consequently an antiserum will contain predominantlyIgs of this class.

Immunoglobulins are sometimes digested enzymatically to produce frag-ments that are still reactive with the antigen. Pepsin digests the constant partof the heavy chain below the 'hinge' region leaving a dimeric fragment calledF(ab')2 (see Figure 1). Papain creates two monomeric fragments, Fab, plusthe constant region of the heavy chains, Fc. Fab fragments are sometimesused in place of the whole Ig.

2.2 Polyclonal antiseraTo raise an antiserum, a group of animals are injected with the purified antigentogether with a non-specific stimulant of the immune response (an adjuvant).A protocol can be found in ref. 6. If the immunization is successful, the antigenwill have stimulated a variety of lymphocytes, each of which will have under-gone several divisions to produce a clone of antibody-producing plasma cells.For this reason, the term 'polyclonal' antiserum is sometimes used.

The site on an antigen with which an Ig reacts is called an epitope. An antigenmay contain several epitopes and an antiserum will contain Igs directedagainst each of them. Furthermore different Igs directed against the sameepitope may have different binding affinities and also may see a slightly differ-ent part of the epitope. An antiserum will therefore contain a variety of Igsreactive with the antigen but with different specificities and affinities. Con-sequently, two antisera are never identical.

Apart from a high concentration of antibodies directed against the appro-priate antigen, an antiserum will also contain other Igs that were presentbefore immunization and may contain antibodies reactive with impurities inthe original preparation of antigen. These Igs may give undesired reactionsrequiring their removal.

2.3 Monoclonal antibodiesMany of these problems are avoided by the use of monoclonal antibodies.These are made by a cloned line of cells that produce a single Ig. The cells are

187


derived by fusing spleen cells from an immunized animal with a line of drug-sensitive myeloma cells. After the fusion, the cells are incubated in the pres-ence of the drug. The only cells to grow will be hybrids resulting from a fusionof a normal spleen cell (which is drug-resistant) and a myeloma cell (whichbrings immortality to the hybrid). The hybrids are cloned and clones pro-ducing specific antibodies are selected and recloned. The resulting cell lines(called hybridomas) each produce indefinitely a single Ig. A full description ofthese methods can be found in ref. 7 or in another volume in this series, Anti-bodies: a practical approach (ed. D. Catty), Vol. 1.

2.4 Purific ation of antibodiesSometimes it is necessary to purify an antibody before attaching a label to it.This is comparatively easy if a monoclonal antibody produced in the super-natant of a hybridoma culture is used. An ammonium sulfate precipitationfollowed by exclusion chromatography would suffice. If a monoclonal anti-body from an ascitic fluid or a polyclonal antiserum is used, the specific anti-body should be separated from the other Igs. If the original antigen isavailable, the purification is best performed by affinity chromatography.

2.5 Specificity of antibody reactionsAn important property of an Ig is the affinity with which it binds to its antigen.The concentration needed to produce a desired end-point clearly dependson this affinity. The 'strength' of a polyclonal antiserum or a preparation ofmonoclonal antibodies is directly related to the concentration of antibodymultiplied by its affinity constant; the higher the affinity, the greater the work-ing dilution. For the reasons given below, antibodies of high affinity generallyshow greater specificity.

Antibodies are used as reagents because of their high specificity. Howeverit is important to realize that they can also give rise to non-specific reactions.These can have three causes.

(a) From impurity antibodies. This only arises with a polyclonal antiserum andshould be detected by the use of appropriate controls (see Section 7). It isless likely to be a problem if the antiserum can be used at a high dilution.

(b) From cross-reactions. These arise if two molecules have similar, but differ-ent, structure. Some antibodies may recognize both molecules but oftenthe undesired reaction will be of lower affinity and will only be a problemif the antibody is used at too high a concentration. This emphasizes thedesirability of using antibodies of high affinity.

(c) From two molecules sharing the same epitope. This possibility will not berevealed by the usual controls and would normally only be shown by adetailed immunochemical study.

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Preparations of monoclonal antibodies should not contain any impurityantibodies. However they are specific for an epitope and could give a mislead-ing result if this epitope was found on more than one protein.

2.6 Storage of antibodiesFor antibodies and antisera obtained commercially, the supplier's instructionsshould be followed. Other reagents should be stored without first dilutingthem. It is advisable to aliquot them into suitable quantities and to store thealiquots frozen. Once an aliquot is thawed it should not be refrozen, as thaw-ing and freezing will denature Igs.

If an antibody is used rarely, it may be stored in amounts sufficient for oneexperimental run and an aliquot thawed when necessary. If this involvesmicrolitre quantities of reagent, the aliquot should be covered with a smallquantity of glycerol before freezing to prevent it freeze-drying.

If a reagent is in regular use, it may be stored in larger aliquots. We havefound that most antisera have a shelf-life at 4°C of at least a month, some con-siderably longer. For such reagents we regularly make them 0.01 % in sodiumazide to prevent bacterial contamination. If reagents are ever contaminated,they should be immediately discarded.

3. Effect of tissue processing on antigensBefore applying an antibody, a section of tissue must be prepared. Beforedeciding how to handle a tissue the effect of any processing on the antigen ofinterest must be considered.

Conventionally, when sections are to be stained with haematoxylin andeosin, tissue is fixed, often in formalin, and embedded in paraffin wax. Aftersections have been cut, the wax must be removed by immersion in xylene, or asimilar solvent, and brought to water through ethanol. This treatment yieldssections of high quality. It also alters the proteins in a tissue so that many anti-gens no longer react with the appropriate antibody. In this case, alternativemethods of fixation and processing may be tried but frequently sections mustbe cut from frozen tissue.

3.1 Choosing conditions for processingWhen using a new antibody, it is important that the optimum conditions forfixation and processing are determined. While these can only be establishedempirically, it is possible to lay down some guide-lines.

A set of fixatives is chosen. We would recommend formalin, methacarn,90% ethanol, Bouin's, chloroform:acetone, and formolxalcium followed bychloroform:acetone. These fixatives are then tested on a set of frozen sections.Several frozen sections are cut from the tissue that contains the antigen ofinterest and one of the sections immersed in each of the selected fixatives for

189


5 min. The sections are stained with the antibody using the selected method andthe result read. If all the fixatives chosen appear to destroy the antigen, theexperiment is repeated using more gentle fixatives (e.g. paraformaldehyde:lysine:periodate). Occasionally, the staining must be carried out without priorfixation; this will give sections with poor morphology.

Recipes for various fixatives, including those mentioned above, with someindications of their use are given in Protocol 1.

Protocol 1. Recipes for some common fixatives

A. Fixatives generally used on tissue subsequently processed into blocksof paraffin wax

1. 10% formol:saline. 100 ml 40% formaldehyde, 9 g NaCI in 900 mlwater.

2. Methacarn. 60% methanol, 30% chloroform, 10% glacial acetic acid.Tissues are usually fixed at room temperature overnight and thentransferred to 70% alcohol.

3. Modified methacarn. Use inhibisol in place of chloroform.

4. Carnoy's fluid. 60 ml ethanol, 30 ml chloroform, 10 ml acetic acid.

5. Bouin's fluid. 75 ml saturated picric acid, 25 ml 40% formaldehyde,5 ml acetic acid.

6. B5. 60 g mercuric chloride, 21 g sodium acetate trihydrate in 900 mlwater. Add 100 ml 40% formaldehyde before use. When using this fix-ative, after sections have been cut, mercury must be removed beforeperforming an immunohistochemical stain. Take sections to water.Immerse for 5 min in Lugol's iodine.a Wash in water. Immerse for afew seconds in 5% (w/v) sodium thiosulfate. Wash in water.

B. The following are usually used on frozen sections

1. Formohcalcium. 100 ml 40% formaldehyde, 100 ml 1 M CaCI2, 800 mlwater, plus a few chips of marble (or CaCO3). Store at 4°C. Fixation isfollowed by a further fixation in chloroform:acetone.

2. Chloroform:acetone (50:50, v/v). 5 min fixation at 4°C. Often used to fixfrozen sections prior to using monoclonal antibodies to distinguishlymphocyte phenotypes.

3. Ethanohacetic acid. 95% ethanol, 5% glacial acetic acid. 1 min at 4°Cfor frozen sections.

4. Ethanol. Various percentages of ethanol at 4°C or room temperaturefor a predetermined time.

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5. Periodate:lysine:paraformaldehyde.

(a) Prepare a 3.6% (w/v) solution of paraformaldehyde by dissolv-ing 2 g in 0.14 M sodium dihydrogen phosphate, 0.11 M NaOHat 70°C.

(b) Filter, cool, and add 2.5 ml 1 M HCI.

(c) Prepare a solution of lysine by adjusting the pH of a 0.2 M solutionof lysine-HCI to 7.4 by addition of 0.1 M dibasic sodium phosphate.

(d) Dilute to 0.1 M lysine by addition of 0.1 M phosphate buffer pH 7.4.

(e) Just before use mix one part of paraformaldehyde solution tothree parts lysine solution and add solid sodium periodate to aconcentration of 10 mM.

This is a gentle fixative that is suitable for labile antigens such as theH2 antigen in mouse tissues.

"Lugol's iodine. 2 g Kl, 1 g iodine in 100 ml water.

If the antigen survives one or more of the fixatives, tissue is fixed in theoptimum fixative and embedded in paraffin wax. Sections are cut and stained.If the processing has destroyed the antigen, this is repeated using a paraffinwax with a low melting point (45 °C as opposed to 58°C). At this stage itshould be possible to select the optimum conditions for a particular antibody.

The antigens found on the surfaces of lymphoid cells, such as the histo-compatibility antigens and the subset-specific markers, are often unstable.Some care must be taken to preserve their integrity. To demonstrate thenecessary steps, a procedure used to prepare sections for staining labilemarkers on the surface of lymphocytes in human tissue is given in Protocol 2.

Protocol 2. Preparation of sections for staining for lymphocyticmarkers

Equipment and reagents• Microtome mounted in a cryostat • Formokcalcium (see Protocol 1)• Glass microscope slides • Acetone• Slices of cork • Chloroform:acetone (50:50, v/v)• OCT embedding compound • Phosphate-buffered saline (PBS)• Isopentane (Aldrich Chemical Co.)

A. Freezing the tissue

1. Cover small pieces of tissue (maximum size 3 x 3 x 15 mm) with OCTembedding compound on a slice of cork.

2. Freeze in isopentane pre-cooled in liquid nitrogen.

3. Store the tissue and cork in plastic ampoules in liquid nitrogen.

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B. Cutting sections

1. Cut sections, 8 um thick, on a microtome mounted in a cryostat andmount as usual on glass slides.

2. Dry sections at 37°C for 1 h. They may now be used immediately orstored.

3. To store sections, wrap them in plastic film (the type sold for 'cling'wrapping food) and store at -20°C. Before use, bring to room tempera-ture and remove film.

C. Fixation

1. 5 min in formol:calcium.

2. Dip in cold acetone.

3. 5 min in chlorofornracetone (50:50, v/v) at -20°C.

4. Dip in cold acetone.

5. Wash twice in PBS.

It should be noted that the various epitopes on an antigen might be affecteddifferently by a fixative. If several monoclonal antibodies to the same antigenare available, each should be optimized separately.

Sections of calcified tissue, such as a tumour metastasized to bone, are nor-mally decalcified with 5% formic acid or 0.5 M EDTA before histologicalstaining. If the antigen has survived fixation in formol:saline, it will usuallyalso survive the process of decalcification.

3.2 Revealing hidden antigensSometimes treatment of a section of fixed tissue with a either heat in a micro-wave oven or with a proteolytic enzyme will 'reveal' an apparently destroyedantigen. The mechanism by which these procedures permit a previously in-hibited reaction of one of the proteins with an antibody is not properly under-stood. Possibly, after fixation, the epitope in question is unaffected butsurrounded by a matrix of cross-linked proteins whose removal allows theantibody access.

The use of a proteolytic enzyme adds to the procedure an extra variablethat is difficult to control precisely. As a general practice we would not recom-mend it. However, in diagnostic pathology, sometimes the only availabletissue has already been fixed in formalin and embedded in paraffin wax andthese may not be the optimal conditions for a particular antigen. In thesecircumstances there is little choice but to try this approach. A suitable recipeis given in Protocol 3. A procedure for revealing antigens using treatment in amicrowave oven (8) is given in Protocol 4.

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Protocol 3. A method for treatment of sections with pronase

Reagents• Phosphate-buffered saline • Pronase

Method

This is used on sections from fixed tissue prior to applying the first anti-body.

1. Dewax the section and take it through alcohol to water.

2. Incubate the section in PBS at 37°C for 5 min.

3. Incubate the section in PBS, 50 ug/ml pronase at 37°C for 20 min.

4. Wash in running tap-water for 5 min.

5. Wash twice in PBS.

6. Apply antisera according to the desired protocol.After this treatment, the sections are very fragile and must be handledwith care.

Protocol 4. A method for the treatment of sections in amicrowave oven

Equipment and reagents• Domestic microwave oven equipped with a • Citrate buffer: dissolve 2.1 g citric acid in 1

temperature probe litre distilled water, add 26.5 ml 1 M NaOH,• 3-aminopropyl triethoxysilane adjust to pH 6

Method

1. Cut 5 p.m sections and mount them on slides coated with aminoalkylsilane (see Section 6.9.1).

2. Dewax sections, take them down to water, and place the sections in aslide holder.

3. Pour 500 ml citrate buffer into a plastic container with a hole cut in thelid to take the temperature probe of the microwave oven. Heat thebuffer to 90°C.

4. Place slides in the pre-warmed citrate buffer and heat in themicrowave oven at 90°C for 10 min.

5. Remove slides from the oven and leave to cool for 15 min.

6. Wash sections in water and continue as for the chosen method.

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The effect is demonstrated in Figure 2 which shows a stain for glial fibrillaryacidic protein on a section of formalin fixed human cerebellum embedded inparaffin wax using an indirect method. In Figure 2b the section was pre-treatedwith pronase according to the protocol in Protocol 3; Figure 2a was untreated.The primary antibody was a rabbit polyclonal antiserum raised by Mr N.Bradley (Institute of Cancer Research) and used at a dilution of 1/100. The sec-ondary antibody was a peroxidase-conjugated swine anti-rabbit (Dako) used ata dilution of 1/100. Colour was developed using diaminobenzidine (DAB), thesection counterstained with Mayer's haemalum (see Section 6.9.2), and the cov-erslip mounted in DPX (see Section 6.9.3). The antibody picks out the astro-cytes, the fine fibrils are the cellular processes delineated by the stain. Thestronger staining on the section pre-treated with pronase can be seen.

4. Choice of labelThe labels used to visualize an antibody fall into three main classes: fluores-cent, enzymatic, and gold with silver enhancement. It is also possible to use aradioactive label followed by autoradiography. This is not a normal immuno-histochemical procedure and the technique will not be discussed here.

4.1 Fluorescent labelsAn ideal fluorescent label has a high quantum yield, good separation betweenthe wavelengths of excitation and emission, a wavelength of maximal absorptionclose to a strong line from a mercury arc lamp (used for fluorescence micro-scopy), and an emission wavelength suitable for photographic film and thehuman eye. In practice, the two substances that are in common use for fluor-escent microscopy are fluorescein and rhodamine which can both be attached toprotein by reaction of lysine residues with the isothiocyanate. Phycoerythrin, afluorescent protein found in red algae, is a popular label for flow cytometry andconsequently a wide range of antibodies labelled with this protein is available.Filters designed for use with rhodamine are also suitable for phycoerythrin.

The advantage of using a fluorescent label is its speed. Once the slide hasbeen incubated with labelled antibody, the coverslip can be mounted and theresult read. The disadvantages are the need for specialist equipment and thatone has literally to work in the dark. The architecture of the tissue and the cel-lular morphology are not revealed. Although the earliest immunohistochem-istry employed antibodies labelled with fluorescein, this method is beingsuperseded by methods employing enzymes.

4.2 Enzymatic labelsWhen an enzyme is used as a label, it is visualized by means of a reaction thatgives an insoluble coloured product. An ideal enzyme has a low molecularweight (for ease of attachment to Ig) and a high turnover number (to give a

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5: Immunohistochfiirtintry

Figure 2. Human cerebellum stained for glial fibrillary acidic protein showing the effect ofpre-treatment with a protease, (a) Untreated, (b) Pre-treated with pronase. The bar repre-sents 30 um.

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high yield of product), is absent from normal tissue, and can be used to give aproduct which is insoluble in water, ethanol, and xylene (so that the coverslipcan be mounted conventionally).

4.2.1 Horseradish peroxidaseHorseradish peroxidase (HRP) was the first such enzyme to be used andremains a popular choice. It fulfils the above criteria except that it is found innormal tissue, particularly granulocytes, erythrocytes, and cells of the myeloidseries. Usually it is necessary to block the activity of enzyme endogenous tothe tissue. The substrate is hydrogen peroxide and the product oxidizes achromogen. It is commonly used with DAB that gives a brown precipitate atthe site of reaction. An advantage of this stain is that, for all practical pur-poses, it is permanent. Care must be taken not to confuse the reaction productwith endogenous brown pigment.

4.2.2 Alkaline phosphataseThe substrate for this enzyme is usually a naphthol phosphate with a diazo-nium salt. The phosphatase releases the naphthol that couples with the diazo-nium salt to form a precipitate. Used in conjunction with Fast Red it gives ared precipitate that dissolves in ethanol and xylene so that the coverslip mustbe mounted in an aqueous medium. Alkaline phosphatase is found in severaltissues including bone marrow, breast, endothelium, kidney, placenta, andintestine. That found in the intestine is a different isoenzyme and is the morerobust, surviving many procedures for processing which destroy the enzymeat other sites. The red colour catches the eye and we have found this label par-ticularly useful when trying to identify rare cells (e.g. micrometastases in bonemarrow smears) (9). It has the disadvantage that the reaction product fadesover a period of months.

4.2.3 Glucose oxidaseGlucose oxidase is found in bacteria and is absent from mammalian tissue.The substrate is oxidized in the presence of a tetrazolium salt and the hydrogenacceptor, phenazine methosulfate. Upon reduction, the tetrazolium forms acoloured precipitate that, if the nitroblue derivative is used, is blue. If usedwhen labelling two antigens on the same slide, it makes a pleasing contrastwith the product from alkaline phosphatase. It is necessary to use MethylGreen if a counterstain of the nuclei is required.

4.2.4 GalactosidaseThe enzyme B-galactosidase is extracted from Escherichia coll and is readilyconjugated to other proteins. The optimal pH for the bacterial enzyme (7-7.5)differs from that of human 3-galactosidase (5-5.6) so that, if the correct bufferis used, there is no need to block the endogenous enzyme. Furthermore, thelatter will be inactivated by heating above 55 °C so that tissue embedded inparaffin wax will contain no active enzyme.

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4.2.5 Conjugating enzymes to antibodiesSeveral manufacturers now produce a range of conjugated second antibodiesof high quality. Producing conjugates in the laboratory is not recommended ifcommercial reagents are available.

4.3 Colloidal goldThis technique employs colloidal gold particles onto which an antibody hasbeen absorbed. It was originally developed for electron microscopy where ithas the advantage that gold is electron dense and that different antibodiesmay be labelled with gold particles of different sizes, enabling two or threeantigens to be localized simultaneously. Used in light microscopy, the goldparticles have to be visualized by a silver precipitation. The method hasproved to be more sensitive than those employing enzymatic labels.

Since immunogold is usually used in an indirect method and several manu-facturers produce suitable reagents, these are usually best purchased. Strepta-vidin absorbed onto colloidal gold is also obtainable and can be used inconjunction with biotinylated antibodies.

The pH, particle size, ionic concentration, and the concentration of proteinaffect the amount of protein absorbed onto the surface of colloidal gold parti-cles. The first of these, pH, is critically important and needs to be close to theisoelectric point of the protein being absorbed. Methods for absorbing pro-teins onto gold are described in the article by J. Roth (ref. 2, Vol. 2), and themanufacturer, Janssen Pharmaceutica, has produced an excellent bookletgiving detailed recipes for different types of protein.

4.4 Selecting a labelThere is no right or wrong choice of label. Selection should be guided by thetissue to be studied. Alkaline phosphatase would probably be a poor choice ifa study is to be made of the gut since there is a high concentration of theenzyme in this tissue. Peroxidase is usually best avoided if the tissue containslarge amounts of endogenous peroxidase or brown pigment (which can beconfused by the colour produced by a commonly used substrate), particularlyif the antigen is likely to be affected by the procedures necessary to eliminatethese. Another important factor is the availability of labelled reagent, sincefor most applications it is usually easier and cheaper to buy rather than makelabelled antibody. The ultimate choice may come down to the personalpreference of the individual worker.

5. Methods of applicationThe simplest method of detecting an antigen in a tissue section is to apply alabelled antibody (the so-called direct method). More commonly the primary

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antibody is left unlabelled and the label is attached to a different reagent thatis then used to detect the primary antibody. For example, if the primary anti-body is a mouse IgG, it may be detected by a labelled goat anti-mouse IgGantibody (the indirect method).

Of the methods described below, the direct method gives the fastest result.The indirect method combines ease of use with acceptable sensitivity, whilethe enzyme-anti-enzyme and immunogold methods are the most sensitive.As in many things, there is no one 'correct' method and the choice is oftengoverned by the personal preferences of the investigator.

5.1 The direct methodThe label is attached directly to the antibody (Figure 3d). To do this, the Igmust first be purified. The advantage of the method is its speed—it has onlyone step. The disadvantages are that it is less sensitive than other methodsand that each antibody has to be labelled separately.

5.2 The indirect methodA second antibody is raised to the Igs of the species from which the antibodyof interest was obtained. Sections are incubated with the first antibody,washed, and then incubated with the labelled second antibody (Figure 3b).

The major advantage is that, for a series of first antibodies, only one prepa-ration of labelled second antibody is needed. This creates less work and also ismore economical with the primary antibody since some reagent is always lostduring a chemical procedure. The indirect method is also more sensitive thanthe direct method because more than one second antibody molecule can reactwith each first antibody.

Figure 3. The (a) direct and (b) indirect methods of visualizing the reaction site of an anti-body on a tissue section. 1, primary antibody; 2, labelled second antibody.

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In a particularly sensitive variant of the indirect method, the second anti-body is absorbed onto particles of colloidal gold (see Section 4.3).

5.3 Enzyme-anti-enzyme methodsThis is a variation of the indirect method which is used to increase sensitivity.The label used in immunohistochemistry is frequently an enzyme. For ease ofdescription, it is assumed that the first antibody is a mouse monoclonal. Amouse antibody to the enzyme is also required. A further requirement is anunlabelled antibody (e.g. raised in a rabbit) to mouse IgG.

As it outlines in Figure 4, the section is incubated in the first (mouse) anti-body, washed, and then incubated in an excess of rabbit anti-mouse IgGserum and washed again. A solution of complexes of enzyme-anti-enzyme isprepared by adding mouse antibodies to a solution of enzyme. This is addedto the slide and, after incubation and washing, a colour reaction for theenzyme performed.

Because Igs are dimers, they can react with two separate molecules of anti-gen. If an enzyme carries more than one epitope and the enzyme and antibodyare mixed in the correct concentrations, a cross-linked network of enzymeand antibody will be formed. The purpose of the rabbit anti-mouse IgG serumis to link the enzyme-anti-enzyme complexes to the first antibody.

The advantage of this method is its greater sensitivity since even moremolecules of label can be added to each molecule of first antibody. It alsoavoids having to link covalently an enzyme to the second antibody. The dis-advantages are the need for an additional reagent (the anti-enzyme antibodies)and the additional time required because of the extra step in the sequence ofreactions.

If the enzyme used is HRP the method is called PAP (peroxidase-anti-

Figure 4. The enzyme-anti-enzyme method. 1, primary antibody; 2, second (linking) anti-body; 3, enzyme-anti-enzyme complex. Note that the primary and the anti-enzymeantibodies must be raised in the same species.

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peroxidase); alkaline phosphatase-anti-alkaline phosphatasc (APAAP) isalso frequently used,

Figure 5 shows a human iymph node stained for the leucocyte commonantigen (CD45) using the indirect method with alkaline phosphatase (Figure5a) and the APAAP method (Figure 5b). Tissue was fixed in modified metha-carn and embedded in paraffin wax. The primary antibody was a murinemonoclonal at a dilution of 1/200. The secondary antibody in Figure 5a was arabbit anti-mouse alkaline phosphatase used at a dilution of 1/200. In Figure5b rabbit anti-mouse Ig was applied at 1/20 followed by mouse APAAP at1/100. The reagents were obtained from Dako. The colour was developedusing Fast Red TR, the counterstain was Mayer's haemalum (Section 6,9.2),and the coverslip mounted in glycerine jelly (Section 6.9.3). The stain demons-trates that the antibody reacted with the surface of the lymphocytes and acomparison of Figure 5a and 5b demonstrates the greater sensitivity of theAPAAP as compared to the simple indirect method.

5.4 Systems using biotin-avidinAvidin, a protein extracted from egg white, has four binding sites of highaffini ty for biotin which is found is liver. Biotin can be covaicntly bound toeither the first or second antibody which is then visualized using labelled

Figure 5. Human lymph node stained for leucocyte common antigen using (a) the indi-rect and (b) the APAAP method. The bar represents 200 (j.m,

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Figure 6. The biotin-avidin method. 1, primary antibody; 2, biotinylated second antibody;3, labelled avidin.

avidin (Figure 6). Avidin has an isoelectric point close to 10 and is positivelycharged in neutral buffers. It is therefore likely to bind negatively chargedmolecules in a tissue. Streptavidin, isolated from Streptomyces avidinii, alsohas four binding sites for biotin but has an isoelectric point close to 7.

For enzyme labelling, rather than attach an enzyme directly to avidin orStreptavidin, the enzyme may be biotinylated and unlabelled avidin used as abridge. For increased sensitivity, this system may be used in a manner analogousto the enzyme-anti-enzyme technique. Complexes of biotinylated enzyme-avidin are preformed and applied in place of the labelled avidin—the so-called ABC method (avidin-biotinylated peroxidase complex). It is claimedthat larger complexes can be created than in the enzyme-anti-enzyme methodthereby giving greater sensitivity.

Because of the presence of biotin in liver, particular care should be exercisedif avidin or Streptavidin are used on sections of this tissue.

5.5 Other methodsThe avidin-biotin system can be mimicked by attaching a small molecule(hapten) to the first antibody and using a labelled antibody to the hapten.Common haptens are dinitrophenol (DNP) and arsinilate. This method canbe useful when labelling a section with two antibodies from the same species(see Section 8).

The methods above may be used in a variety of combinations. In attemptsto increase sensitivity, antibodies may be piled on antibodies. Except in

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special circumstances, this is generally neither necessary nor desirable. If anantigen has apparently been destroyed in fixed tissue, its presence may berevealed by using a highly sensitive method. However, the increased sensitiv-ity will also show weak cross-reactions and amplify any slight non-specificstaining. Whenever possible, it is preferable to select the correct conditionsfor processing a tissue.

6. Experimental methodsWork sheets for the different methods are presented below. They may varyin detail from those of other authors. There is no one correct method andvariation can be introduced as long as certain guide-lines are followed.

In the following methods, all incubations and treatments are at room tem-perature unless otherwise stated. In many laboratories, this can vary from18-24 °C and this will lead to considerable variation in the rate of reaction. Ifstandardization is desired, and particularly if quantitative measurements areto be made, the temperature should be controlled. If a cold room is used, thetimes of incubation should be lengthened.

It has been reported that times of incubation with antibody solutions can bereduced from 1 h to 1 min by use of a microwave oven (10). This relies on thespeed of the antibody-antigen reactions at the high temperatures induced bylocalized heating by the microwaves.

6.1 A general methodA generalized method for applying antibodies to a section is given in Protocol5. This is for an indirect method but can be adapted for all the other methodsabove. It is to be used together with the detailed protocols for each type oflabel in the methods listed in the rest of this section.

Protocol 5. The indirect method

Equipment and reagents• Moist chamber (see Section 11) • Phosphate-buffered saline (PBS)• PBS, 5% serum: the serum being obtained • PBS, 0.5% bovine serum albumin (BSA)

from the same species as the second anti- . PBS, 0.01% detergent (Brij or Tween 80)body

Method

1. Bring the section to water or buffer.

2. When using an enzyme conjugate, if necessary block endogenousenzyme.

3. Rinse in PBS and wipe any excess from the slide with a paper tissue.This ensures that the antiserum is not diluted on the slide.

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4. Place 100 ul of the first antibody, diluted appropriately,a over thesection. Incubate for 1 h at room temperature in a moist chamber.

5. Wash the section with PBS, 0.5% BSA.

6. Wash several times in PBS, 0.01% detergent.

7. Wash with PBS and wipe excess from the slide.

8. Place 100 ul of the second (conjugated) antibody, diluted appropri-ately, over the section. Incubate for 1 h in a moist chamber at roomtemperature.

9. Repeat steps 5 and 6.

10. If using an enzymatic label, wash the slide in an appropriate bufferand develop the colour. Mount the coverslip.

' Dilute antibodies in either PBS, 0.5% BSA or preferably, in PBS, 5% serum.

The purpose of most of the additives to the buffers is to prevent Igs stickingnon-specifically to the section and to remove the last traces of unreacted Igafter incubation. Protein is added to prevent the antibody absorbing onto thesurface of the section and the addition of a small amount of detergent isintended to reduce hydrophobic interactions between Igs and proteins in thesection.

If large numbers of sections are being stained, for washing, they can beplaced in racks and gently agitated in a staining trough. If there are only a fewsections, solutions for washing may be kept in wash bottles. When a section iswashed, the slide is held at a slant and a stream of solution directed just abovethe section. The sections will be washed several times and care must be takennot to wash the section off the slide. This also emphasizes the need to preparesections of high quality.

6.2 Choosing the correct dilution of antibodyWhen using a new antibody, a number of sections should be cut from a tissueknown to contain the antigen. They should be stained using a set of dilutionswhich should be in geometric, not arithmetic, progression (e.g. 1:10, 1:20, 1:40,etc.). It is best to start with large steps (e.g. in fives) to find the approximaterange and then to repeat the experiment using doubling dilutions.

The optimal dilution is that which just stains a section close to the maxi-mum strength. With some antisera, at high concentrations, non-specific stain-ing of the section may be observed. In this case, a dilution has to be sought atwhich the non-specific staining has been diluted out while the specific stain isstill acceptably strong. This should not be a problem when using a monoclonalantibody.

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6.3 Fluorescent labelsSome fixatives (e.g. glutaraldehyde) make a tissue autofluorescent. Thisshould be checked before starting. After following the procedure in Protocol 5,coverslips should be mounted in an aqueous, non-fluorescent mountant (suchas the Fluorescence Mounting Medium from Dako Ltd.) or glycerol:40%formaldehyde (9:1, v/v). Another mountant can be made up as follows.

(a) To 3 g analytical grade glycerol add 1.2 g poly vinyl alcohol (PVA) and stir.(b) When the PVA and glycerol have mixed completely, add 3 ml water, stir,

and leave for 4 h at room temperature.(c) Add 6 ml 0.1 M Tris-HCl buffer pH 8.5, and keep at 50°C with occasional

agitation until the PVA has dissolved.

6.4 PeroxidaseThe chromogens used with peroxidase often produce a brown colour thatmight be confused with brown pigment in the section, for example, that foundin red blood cells in tissue fixed in formol:saline. It is therefore often neces-sary to bleach the section before starting any other procedure. This is done byimmersing the slide in H2O2 (e.g. 7.5%) for 5 min and then washing well inwater. A check should be made to see if this has a deleterious effect on theantigen being studied.

6.4.1 Blocking endogenous enzymeBefore placing antibody on the section, incubate the slides in 2.3% periodicacid for 5 min, wash in water, rinse in 0.03% freshly prepared potassium boro-hydride, and wash in water. Another method often used is to incubate theslides for 30 min in 0.3% H2O2 in methanol. If these treatments destroy theantigen, an alternative is to incubate the slides in PBS, 0.1% phenylhydrazinefor 5 min.

6.4.2 Chromogens for peroxidaseFrom the large number of possible chromogens for use with peroxidase, onlyabout five have found general use in immunohistochemistry. Their recipes aregiven below.

i. Diaminobenzidine (DAB) with or without enhancementThis is the most commonly used chromogen. It gives a brown precipitatethat is insoluble in water, ethanol, and xylene. DAB has been suspected as acarcinogen and should be handled with care.

(a) Just before use dissolve 100 mg DAB in 100 ml 0.1 M Tris-HCl bufferpH 7.2.

(b) Add 100 ml water containing 70 ul 30% H2O2.

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(c) Immerse sections for 5 min. Wash thoroughly in water.(d) Counterstain in Mayer's haemalum and mount coverslips in DPX (see

Section 6.9).

It is possible to intensify the stain using either imidazole or silver. For theformer, make the DAB solution above 0.01 M in imidazole before use.

For silver enhancement, a method is given in Protocol 6. The result of asilver enhancement is shown in Figure 7. Sections are of human colon fixed informalin and embedded in paraffin wax. They were stained for carcino-embryonic antigen (CEA) by the indirect method. The primary antibody wasrabbit anti-CEA raised by one of the authors (M. G. O.) and used at a dilutionof 1/4000. Secondary antibody and colour development as Figure 2. In Figure 7athere was no further treatment; in Figure 7b the colour was enhanced withsilver. CEA is located on and in the epithelial cells. The staining is barelyvisible at this dilution of primary antibody without enhancement but is clearlydemonstrated after the silver reaction.

Protocol 6. Silver enhancement of a peroxidase/diaminobenzidinestain

Reagents2.5 mM gold chloride pH 2.3100 mM sodium sulfide pH 7Acetic acidSodium carbonateAmmonium nitrateSilver nitrateDodeca-tungstosililic acidMayer's haemalum

' Silver reagent. Make up the following solu-tions by dissolving each given quantity in 100ml twice distilled water. Solution A: 5.08 gsodium carbonate; B1: 0.83 g ammoniumnitrate; B2: 0.82 g silver nitrate; B3: 3.97 gdodeca-tungstosililic acid. Add 1 ml solutionsB1, B2, and B3 to 1 ml water. Add 5 ul 40%(v/v) formaldehyde solution. Add this solutionto 4 ml solution A with vigorous mixing.

Method

The section is first stained using a suitable method with a peroxidaselabelled antibody and the colour developed with DAB. The colour maythen be enhanced using the method below.

1. Wash the sections in distilled water.2. Immerse for 5 min in 2.5 mM gold chloride pH 2.3. Wash in distilled

water.3. Immerse for 5 min in 0.1 M sodium sulfide pH 7. Wash in distilled water.4. Immerse for 2-6 min in the silver reagent. Wash thoroughly in distilled

water leaving the slide in water for 10 min between washes.5. Immerse in 1% (v/v) acetic acid for 15 min changing the acid once

during this time. Wash in water.6. Counterstain in Mayer's haemalum and mount.

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Figure 7. Human colon stained for CEA showing the effect of silver enhancement on theproduct of the peroxidase reaction. The bar represents 50 um,

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ii. Hanker-Yates reagentThis consists of one part p-phenylenediamine-HCl to one part (w/w) pyro-catechol. It gives a blackish/brown precipitate insoluble in water, ethanol, andxylene.

(a) Just before use dissolve 150 mg Hanker-Yates reagent in 100 ml 0.1 MTris-HCl buffer pH 7.6, and add 120 ul 30% H2O2.

(b) Incubate sections for 15 min. Wash in water.(c) Counterstain in Mayer's haemalum and mount in DPX.

Hi. 3-amino-9-ethyl carbazoleThis gives a red precipitate soluble in ethanol.

(a) Dissolve 2 mg 3-amino-9-ethyl carbazole in 0.5 ml dimethyl formamide(DMF) in a glass tube.

(b) Add 9.5 ml 0.2 M acetate buffer pH 5.(c) Just before use add 5 ul 30% H2O2. Incubate sections for 15 min.(d) Counterstain in Mayer's haemalum and mount in glycerine jelly.

iv. 4-chloro-l-naphtholThis can be used if a blue colour is required. The precipitate is soluble inxylene. We have not found a suitable nuclear Counterstain since MethylGreen is soluble in water.

(a) Dissolve 20 mg 4-chloro-l-naphthol in 40 ml 20% methanol in Tris-salinepH 7.6, by heating to 50°C.

(b) Before use add 15 ul 30% H2O2. Incubate sections for 10 min.(c) Mount in glycerine jelly.

v. Tetramethyl benzidineThis is an alternative blue stain, insoluble in ethanol and xylene.

(a) Dissolve 5 mg tetramethyl benzidine in 2 ml dimethyl sulfoxide.(b) Add to 50 ml 0.02 M acetate buffer pH 3.3, containing 20 ul 30% H2O2

immediately before use. Incubate sections for 15 min.(c) Counterstain in Methyl Green and mount in DPX.

6.5 Alkaline phosphatase6.5.1 Blocking endogenous enzymeBefore applying the first antibody, immerse the slides in 20% acetic acid for5 min. Wash well in water. This will destroy all alkaline phosphataseactivity including that in the intestine. If it also destroys the antigen, alkaline

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phosphatases other than intestinal can be inhibited by making the substratesolution 1 mM in levamisole (increased to 2 mM for frozen sections of tissuesrich in alkaline phosphatase such as placenta and kidney). This takes advan-tage of the fact that the alkaline phosphatase used for preparing conjugatedantibodies is extracted from calf intestine.

6.5.2 A cautionary notePhosphate buffer may inhibit alkaline phosphatase. If the last antibody hasbeen washed off in phosphate buffer (as recommended in the protocol inProtocol 5), the sections should be washed thoroughly in distilled waterbefore developing the colour.

6.5.3 Chromogens for alkaline phosphatasei. Fast RedThis is the usual chromogen for use with alkaline phosphatase. It gives a redprecipitate soluble in ethanol.

(a) Dissolve 5 mg sodium naphthol AS BI phosphate in a few drops of DMFin a glass tube.

(b) Add to 5 mg Fast Red TR salt in 10 ml veronal acetate buffer pH 9.2.(c) Add levamisole if required. Filter.(d) Incubate slides for 1 h and wash in water.(e) Counterstain with Mayer's haemalum and mount in glycerine jelly.(f) The intensity of the reaction may be increased if the substrate is renewed

after 30 min.

ii. Fast Blue saltThis gives a blue product soluble in ethanol.

(a) Dissolve 5 mg Fast Blue BB salt in 10 ml 0.1 M Tris buffer pH 9, and add5 mg sodium naphthol AS BI phosphate in DMF as above.

(b) Add levamisole if required. Filter.(c) Incubate for 15 min replacing the substrate after each 5 min. Wash in

water.

There are problems finding a counterstain for this dye since haemalumstains the nuclei blue, Methyl Green is soluble in water, while the blue precip-itate is soluble in xylene.

Hi. NewFuchsinThis produces a red precipitate insoluble in ethanol and xylene.

(a) Mix 250 ul 4% New Fuchsin in 2 M HC1 with 250 ul 4% NaNO2 and leaveto stand in the cold for 5 min.

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(b) Add to 40 ml 0.2 M Tris-HCl buffer pH 9, and add 10 mg sodiumnaphthol AS TR phosphate dissolved in 0.2 ml DMF.

(c) Add levamisole if required. Filter. Incubate sections for 10 min.

(d) Counterstain with Mayer's haemalum and mount in DPX.

6.6 Glucose oxidaseThe chromogen usually used with glucose oxidase is nitroblue tetrazoliumthat gives a dark blue precipitate insoluble in ethanol and xylene. It shouldnot be necessary to block endogenous enzyme.

(a) Dissolve 335 mg B-D-glucose and 33.5 mg nitroblue tetrazolium in 50 ml0.05 M Tris buffer pH 8.3.

(b) Heat at 37°C for 1 h in the dark and add 8.3 mg of phenazine metho-sulfate (this compound may be carcinogenic and should be handled withcare).

(c) Incubate sections for 1 h at 37 °C in the dark. Wash in water.

(d) Counterstain in 0.1% Methyl Green and mount in DPX.

6.7 GalactosidaseIf the reagents below are used, the product is blue, stable in ethanol andxylene. It should not be necessary to block the endogenous enzyme.

(a) To 7 ml PBS, 1 mM MgCl2 pH 7, add 0.5 ml 50 mM potassium ferri-cyanide and 0.5 ml 50 mM potassium ferrocyanide.

(b) Add 10 mg 5-bromo-4-chloro-3-indolyl-B-D-galactosidase previously dis-solved in a drop of DMF (this solution may be stored frozen for twomonths).

(c) Incubate sections in the above for 1 h at 37°C. Wash in water.

(d) Counterstain in 0.1% Methyl Green and mount in DPX.

6.8 ImmunogoldThe gold is visualized by silver precipitation by a chemical process similar tothat used to develop photographic film. If the tissue has been fixed in formalinand embedded in paraffin wax, it is necessary to pre-treat the sections withLugol's iodine or some other oxidizing agent (11). The reason for this is notclear since it is not necessary when other conditions of fixation and processingare used. A suitable protocol is given in Protocol 7.

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6.9 Some general procedures6.9.1 Coating the slidesDuring an immunohistochemical stain the slides are washed frequently. It isimportant that the section adheres well to the glass slide. Several differentprocedures are used to coat slides to improve adhesion. Those commonly

210

A. Before adding the first antibody

1. Wash the section in water.

2. Immerse the section for 5 min in Lugol's iodine. Wash in water.

3. Rinse in 2.5% (w/v) sodium thiosulfate in water.

4. Wash in TBS, 1% Triton X-100.

B. Adding the antibody

1. Apply antibodies as described in Protocol 5 substituting Tris-bufferedsaline (TBS) for PBS and using antibody absorbed on immunogold inthe final step.

2. The immunogold method is very sensitive and can bring up any slightbackground staining.

3. Before applying an antibody, it may be necessary to incubate thesection for 10 min in a normal serum (from the same species as thesecond antibody).

C. To develop the stain

1. Wash in water.

2. Incubate in the silver enhancement solution in subdued light (e.g. adark-room safe light) for 40-60 min.

3. Wash in water.

4. Counterstain. Take through ethanol to xylene (or Histoclear) andmount.

Protocol 7. Silver enhancement of colloidal gold

ReagentsLugol's iodine (see Protocol 1, step 6)Sodium thiosulfateTris-buffered salineCitric acidTrisodium citrateGum accaciaSilver lactate

HydroquinoneSilver enhancement solution: 20 ml 1 Mcitrate buffer (1 M citric acid, 0.5 M tri-sodium citrate) pH 3.5; 33 ml 30% gumacacia; 15 ml silver lactate (0.11 g in 15 ml);15 ml hydroquinone (0.85 g in15 ml); 17 mlwater

5; Immunohistochemistry

used (gelatin:formaldehyde, gelatin, albumin, poly-L-lysine, and 3-amino-propyl triethoxysilane) are given in Protocol 8 that also indicates the circum-stances under which they are used. Pre-coated slides can be purchased fromPolySciences (aminoalkyl silane coated called SectionLock) or Sigma (amino-alkyl silane coated called Silane-Prep and poly-lysine coated called Poly-Prepslides).

Protocol 8. Solutions used for coating slides

Reagents• Gelatin• Formaldehyde• Chrome alum• Glacial acetic acid• Ethanol• Egg albumin

Sodium chlorideGlycerineThymolPoly-L-lysine (high molecular weight, Sigma)Acetone3- aminopropyl triethoxysilane

A. Gelatin:formaldehyde (often used for frozen sections)

1. Mix 100 ml 1% gelatin (warm gently to dissolve) and 100 ml 2%formaldehyde.

2. Immerse slides for 3 sec and dry at room temperature.

3. Store at room temperature and use as required.

B. Another gelatin-based adhesive (sometimes used for frozen and softwax sections)

1. Melt 15 g gelatin in 500 ml warm water.

2. Dissolve 1 g chrome alum in 220 ml distilled water.

3. Mix the two and add 70 ml glacial acetic acid and 300 ml 95% ethanol.

4. Store at room temperature.

5. Coat slides as in step 1 above.

C. Albumin (routinely used for ordinary paraffin wax sections)

1. Dissolve 2.5 g egg albumin, 0.25 g NaCI in 50 ml distilled water (warmto 37°C).

2. Add 50 ml glycerine, 0.05 g thymol.

3. Coat slides just before use.

D. Poly-L-lysine

1. Immerse slides in 100 ug/ml poly-L-lysine in water.

2. Dry and store.

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E. 3-aminopropyl triethoxysilane

1. Immerse slides in a 3% solution in either acetone or 95% ethanol.

2. Wash in either acetone or 95% ethanol.

3. Wash thoroughly in water.

4. Dry and store.

6.9.2 CounterstainingIt is general practice to counterstain the nuclei in a section in order to revealthe architecture of the tissue. If the immunohistochemical stain is red, black,or brown, either haematoxylin or Mayer's haemalum is generally used. Weuse the latter. If the immunostain is blue, then Methyl Green is preferred.

i. Mayer's haemalum

(a) Dissolve 1 g haematoxylin in 1 litre of distilled water using gentle heat.(b) Add 50 g aluminium potassium sulfate, heat if necessary.(c) Add 0.2 g sodium iodate, mix well, and leave overnight.(d) Add 1 g citric acid. Mix well.(e) Add 50 g chloral hydrate.(f) Immerse the slide in the haemalum for 5-20 min (depending on the

desired strength of nuclear stain) and wash thoroughly in running tap-water.

(g) Dip in a saturated solution of lithium carbonate that renders the stainblue and wash in tap-water.

ii. Methyl GreenUse a 0.1% solution of Methyl Green in distilled water. The stain will dissolveout in water and the coverslip should be mounted in a non-aqueous mountant.

6.9.3 Mounting the coverslipIf the reaction product is insoluble in ethanol and xylene, the slides arebrought from water through ethanol to xylene. The coverslips are mounted ina natural or synthetic resin such as DPX. If not, they are left in water and thecoverslips mounted in glycerine jelly or a similar water-based mountant.

DPX consists of 10 g distrene 80, 5 ml dibutylphthalate, and 35 ml xylol. It isusually purchased ready made-up.

To make glycerine jelly:

(a) Dissolve 10 g gelatin in 60 ml distilled water using gentle heat.(b) Add 70 ml glycerine and 0.25 g phenol and mix well.

212


(c) Aliquot into 10 ml batches and store in the cold.(d) Before use melt in a water-bath; avoid shaking as this creates air bubbles.

7. Controls and problem solvingThere are two types of problem encountered: the unwanted presence of stainand the unexpected absence of stain. That there is a problem will be revealedby the appropriate controls.

7.1 ControlsTwo types of control are needed—positive and negative. For each antibody, ablock of tissue known to contain the antigen should be selected and a largenumber of sections cut. One of these should be included in every run in orderto monitor the strength of the staining reaction.

Each run should also include an experimental section on which the firstantibody has been omitted. This checks for non-specific staining by thereagents used to detect the primary antibody. If the direct method is beingused, this control would be omitted. If an enzyme stain is being used, a sectionthat is developed for colour only is included to monitor endogenous enzymeactivity.

These controls, although necessary, do not check for non-specific stainingby the primary antibody. When using a polyclonal antiserum, an aliquot ofserum can be absorbed with the original antigen. This should reduce thespecific and reveal non-specific stain. It will not demonstrate the presence ofcross-reacting antigens. It is unnecessary to do this control with every run butit should, if the antigen is available, be performed when first using a new anti-serum and on one or two key sections. This type of control is meaningless witha monoclonal antibody.

The test section itself may act as a control. The distribution of an antigen isprobably known; for example, an antibody against T lymphocytes should notstain epithelial cells. The correct structures should be well stained and otherstructures quite clean. If the 'wrong' cells are stained then non-specific stain-ing can be suspected. In particular, stromal cells and muscle cells tend to givea 'dirty' background stain if the washing procedures are inadequate.

7.2 Problem solvingProblem solving is a matter of applying simple logic. The controls discussedabove should pin-point which part of the procedure is in error. Each stepshould be carefully considered in turn. Frequently the source of the problemis something quite trivial. For example, it is advisable to use slides with endsof frosted glass and to mark the slide clearly in pencil. Without this, it is some-times difficult to recognize one side of the slide from the other and hence stainthe wrong side of the slide.

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There are three types of incorrect staining—under-, over-, and non-specificstaining. No staining at all on the positive control usually suggests that areagent has been inadvertently omitted or the wrong reagent used. If a largenumber of sections have been stained with different antibodies, perhaps theincorrect second antibody has been used on that section (anti-rabbit on a sec-tion stained with a mouse antibody).

Weak staining suggests that one of the more labile reagents has deterio-rated. For example, if a peroxidase stain is in use, it is important that the solu-tion of H2O2 is fresh. Sodium azide is frequently added to buffers to preventbacterial growth. This compound inhibits many enzymes and could cause dif-ficulties if a buffer containing azide has been used during the development ofthe chromogen.

Over-staining is often caused by accidentally diluting one of the reagentsincorrectly. Temperature can occasionally cause a problem. Most routinework is carried out at room temperature, which can vary as much as 10°C in alaboratory without air-conditioning. Enzyme reactions proceed much faster athigher temperatures and a procedure worked out during a chilly day mightover-produce chromogen during a heat wave.

If the volume of antibody solution is insufficient to cover the section prop-erly, any evaporation during incubation will concentrate antibody at theedge of the section. This will cause over-staining. In the extreme, if solutionactually dries onto part of the section, a heavy background stain will result.

Non-specific staining will increase if the slide is over-stained. Apart fromthis, if this is a general problem, attention should be paid to the proceduresused for washing the slides and, in particular, to the protein in the buffer. If aproblem arises with a particular primary antibody, it may help if the section ispre-incubated for 15 min with PBS containing 5% serum from another species(see footnote to Protocol 5). If a particular secondary reagent causes a prob-lem, it should be discarded and another purchased from a different source.

8. Detecting two antigens on the same sectionThe application of two antibodies to the same section requires some care. Ifboth antibodies have been raised in different species, an indirect method maybe used with two differently labelled second antibodies selected to ensure thatthere is no cross-reaction between them. If the two antibodies are from thesame species, as will often be the case when working with murine monoclonalantibodies, it may be possible to carry out a complete immunohistochemicalstain for the first antigen followed by a stain for the second using a differentenzyme. (The formation of the coloured precipitate will often prevent the firstantibody reacting with the second set of reagents.) If this does not work, theneither a direct method should be used with the antibodies each conjugated toa different label or a distinguishing compound must be attached to each anti-

214


body. For example, one antibody could be biotinylated and then detectedusing labelled avidin and the other reacted with dinitrophenol (DNP) anddetected with a labelled anti-DNP.

The correct order of application of the reagents must be established empiric-ally. If antibodies from two different species are used, good results areobtained usually by applying both first antibodies together followed by bothsecond antibodies, and theoretically this should always work. However, some-times better results are obtained if the reagents are applied sequentially.

Enzyme labels are satisfactory if the two antigens are located on separatecells (e.g. when distinguishing different transplantation antigens in chimericmice) or are found in separate cellular compartments (e.g. nucleus and plasmamembrane). If not, then it is difficult to distinguish unequivocally a singly froma doubly labelled cell. In this case, fluorescent labels must be used togetherwith a microscope that allows the operator easily to switch filter combinations(i.e. from those appropriate for fluorescein to those for rhodamine and backagain) while observing a particular cell.

9. Cytological preparationsProcedures for staining cytological preparations are the same as those forsections. The key to achieving good results lies in the method used to preparethe cells. On conventionally prepared smears, there is often protein or mucusoverlying the cells and this can hinder good interaction between cellular anti-gens and antibodies. An excessive number of red blood cells is also undesir-able. It is important to wash the cells, and if necessary remove erythrocytes,before making a smear or centrifuging cells onto a glass slide.

Some methods for the preparation of smears of cells from serous effusions,bone marrow, and cervical scrapes are given in Protocols 9-11. In our hands,the smears were subsequently stained with antibodies to epithelial antigenssuch as epithelial membrane antigen or cytokeratin. The methods might haveto be modified for more labile antigens.

Protocol 9. Preparation of smears from serous effusions

Equipment and reagents• Bench centrifuge • Lymphoprep, density 1.077 (Nyegaard) or. 95% ethanol Histopaque 1077• Carbowax fixative

A. With little contamination from red blood cells

1. Centrifuge the specimen at 300 gfor 5 min.

2. Remove the supernatant and examine the deposit.

3. If the deposit is blood-stained, remove red cells (see below).

215


Protocol 9. Continued4. If the deposit is essentially free of blood, resuspend in 20 ml PBS and

recentrifuge. Repeat.

5. Remove the supernatant and resuspend the cells in as small a volumeof PBS as possible.

6. Place a drop on a clean slide and smear with a second slide as for ablood film. Fix immediately in 95% alcohol and leave for a minimum of1 h.

7. Store in alcohol or spray the smear with Carbowax fixative and storeat-20°C.

B. From blood-stained effusions

1. Wash the centrifuged deposit with 20 ml PBS, and recentrifuge.Remove the supernatant.

2. Mix the centrifuged deposit with 5 ml PBS.

3. Underlay the cell suspension with 10 ml Lymphoprep or Histopaque.

4. Centrifuge at 300 g for 20 min.

5. Remove the layer of nucleated cells at the top of the interface andtransfer to a clean centrifuge tube.

6. Centrifuge at 300 g for 5 min and remove the supernatant.

7. Continue as in part A, step 5.

Protocol 10. Preparation of smears from aspirates of bonemarrow

Equipment and reagents• Bench centrifuge • Lymphoprep, density 1.077 (Nyegaard) or. 50 ml centrifuge tubes Histopaque 1077. Heparin • Sterile PBS

Method

1. Aspirate 1-4 ml marrow using a heparinized syringe and place in a50 ml centrifuge tube containing 1000 U heparin plus 5 ml tissueculture medium.

2. Mix the sample and then make it up to 35 ml with sterile medium.

3. Underlay with 15 ml Lymphoprep.


5. Aspirate the cell layer, transfer to a clean centrifuge tube, and makeup to 20 ml with PBS.

216



7. Aspirate down to 10 ml and make up to 20 ml with sterile PBS.


9. Aspirate down to ~ 0.6 ml, mix well, and transfer to a 1 ml siliconizedconical centrifuge tube.

10. Centrifuge.

11. Aspirate supernatant leaving volume approximately equal to pelletsize.

12. Resuspend very gently and disaggregate the cells by taking up thesuspension into a 20 ul pipette.

13. Prepare thin smears and fix immediately in absolute ethanol. Leavefor at least 30 min. Store at -20°C.

A similar method can be used to prepare nucleated cells fromperipheral blood.

Protocol 11. Preparation of smears from cervical scrapes

Reagents• Cellfix solution: 1 g dithiothrietol dissolved • Dithlothrietol

in 600 ml PBS plus 400 ml ethanol

Method

1. Take a cervical scrape in a conventional manner using a woodenspatula.

2. Break the end from the spatula and drop into 10 ml Cellfix solution.Agitate violently. Remove the spatula. The cells can be stored in thisform at 4°C.

3. Centrifuge the ceils and wash in PBS.

4. Resuspend the cells in the smallest possible volume of PBS. Spread10 ul on a clean microscope slide and allow to air dry.

5. Store the smears at-20°C.

Figure 8 shows a photograph of a smear made from a cervical scrape andstained by the indirect method. The primary antibody was rabbit anti-epithelialmembrane antigen used at a dilution of 1/500; second antibody sheep anti-rabbit Ig conjugated to alkaline phosphatase and used at 1/100. Both reagentswere produced in this laboratory. The colour was developed using Fast RedTR, counterstained in Mayer's haemalum, and the coverslip mounted inglycerine jelly. The normal squamous cell is negative while the neighbouringdyskaryotic cell has both its membrane and cytoplasm stained.

217

Michael G. Ormerod and Susanna F. Imrie

Figure 8. Two cells from a cervical scrape from a lesion diagnosed as cervical intra-epithelial neoplasia, grade 2. The bar represents 10 um.

10. QuantificationImmunohistochemical stains can he quantified using a microscope equippedwith a system for image analysis. For further information, see ref. 12. It isprobably easiest to perform on cytologica! preparations since the cells arc sep-arated and the whole cell can be examined. When undertaking quantitativemeasurements, all the variables, including temperature, should be carefullycontrolled.

11. EquipmentApart from the normal equipment needed to cut tissue sections and to slainsections and cytological smears, the only special piece of equipment needed isa staining tray. This can he quite simple; it is used to keep slides horizontallyin a humid atmosphere. We use trays, 36 cm X 36 cm, .5 cm deep with 2.5 cmhigh cross pieces and a lid, made of polymethylmethacrylate (Perspex, Lucite)(see Figure 9). The tray is levelled on the bench using a spirit-level and byplacing pieces of card appropriately under the edges. The slides are placed onthe cross pieces and a little water is put on the bottom of the tray. With the lidin place, this ensures that the solutions of antibody do not evaporate duringan incubation.

Some protocols require incubations at higher temperatures and it is usefulto have an incubator available. To tit in an incubator, a smaller version of the

218

5; Immunohistochemistry

Figure 9. Photograph of a tray suitable for immunohistochemical staining.

staining tray may be needed. This can easily be devised from a sandwich boxsold in most hardware stores. For incubation at a lower temperature, the traycan be placed in a cold room.

The equipment needed and the methods used to prepare blocks of tissueand to cut sections are well described in standard books of reference (13).

References1. Stcrnberger, S. S, and DC Lellis, R. A. (ed.) (1982). Diagnostic immunohistochem-

istry. Masson Publishing USA Int., New York.2. Bullock, G. R. and Petrusz. P. (ed.) (1982 and 1983). Techniques in immnnocyto-

chemistry, Vols 1 and 2. Academic Press, London.3. Cuello, A. C, (ed.) (1982), Immunohistochemistry, Vol 3. IBRO Handbook Series.

John Wiley and Sons, Chichester.4. Polak, J. M. and van Noorden, S. (ed.) (1983), Immunocytochemistry. John Wright

and Sons, Bristol.5. Polak, J. M. and van Noorden, S, (1987). An introduction to immunocytochem-

istry: current techniques and problems, 2nd edn. R. M, S. Handbook 11, OxfordUniversity Press, Oxford,

6. Johnslone. A. and Thorpe. R. (1982). ImmiiHocheminlry in practice. BlaekwetlScientific, Oxford.

7. Galfre, G. and Milstein, C. (1981). In Methods in enzymology (ed. J. J. Langoneand H. V. Vunakis), Vol. 73, p. 3. Academic Press. New York.

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8. Cuevas, E. C., Bateman, A. C., Wilkins, B. S., Johnson, P. A., Williams, J. H., Lee,A. H. S., et al. (1994). J. Clin. Pathol.,47, 448.

9. Dearnaley, D. P., Ormerod, M. G., Sloane, J. P., Lumley, H., Imrie, S. F., Jones,M., et al. (1981). Br. J. Cancer, 44, 85.

10. Chui, K. Y. (1987). Med. Lab. Sci, 44, 3.11. Holgate, C. S., Jackson, P., Cowen, P. N., and Bird, C. C. (1983). J. Histochem.

Cytochem., 31, 938.12. Read, N. G. and Rhodes, P. C. (1993). In Immunocytochemistry: a practical

approach (ed. J. E. Beesley), pp. 127-49. IRL Press at Oxford University Press,Oxford.

13. Bancroft, J. D. and Stevens, A. (ed.) (1982). Theory and practice of histologicaltechniques, 2nd edn. Churchill Livingstone, Edinburgh.

220

Calcium and pH imaging in livingcells

RICHARD M. PARTON and NICK D. READ

1. IntroductionCalcium and pH imaging, involving the use of ion-sensitive fluorescent dyes,have had a profound impact on our understanding of signal transduction inanimal, plant, and fungal cells. This chapter provides an introduction tothe techniques involved in using the light microscope to image and measureintracellular free calcium and pH within living cells and tissues.

2. Study of calcium and pH in living cells'Second messengers' are intracellular mediators which provide importantlinks in signal-response coupling within cells and act in both signal propaga-tion and amplification (1). The most common and ubiquitous second messen-ger within eukaryotic cells appears to be Ca2+ (2-4). The role of pH hasreceived far less attention as a second messenger. Nevertheless, a variety ofcellular responses to stimuli involve changes in cytoplasmic pH (5-8). In addi-tion, pH homeostasis is important in regulating numerous other aspects of cellmetabolism (9-11).

In order to demonstrate a causal relationship between a change in Ca2+ (orpH) and a cellular response to a specific stimulus, there are several criteriawhich one should aim to establish:

(a) A positive correlation between the response to a stimulus and the changein ion concentration.

(b) The change in ion concentration should precede the response.(c) Prevention of the change in ion concentration using inhibitors or chelators

should inhibit the response to a stimulus.

(d) Artificial changes to the ion concentration (e.g. by using caged probesor ionophores), mimicking the changes which occur upon stimulation,should evoke a comparable cellular response.

6

Richard M. Parton and Nick D. Read

For all of these criteria to be met the concentration of the ion in questionmust be directly measured in living cells and ideally over the whole periodbetween stimulation and the initiation of the response. Furthermore, it isdesirable that the method used to study Ca2+- or pH-mediated signalling pro-vides accurate information on the dynamic spatial and temporal aspects ofchanges in ion concentration, and the magnitude of these changes, as all ofthese features can encode essential signal transduction information (4).Ideally the method of measurement should:

(a) Show high selectivity for the ion of interest over other ions which may bepresent.

(b) Allow accurate and precise quantification of ion concentration. Even verysmall differences in ion concentration may be biologically relevant somethods should be precise enough to measure these differences. The pre-cision of measurement should be of the order of 100 nM Ca2+ and 0.1 pHunit.

(c) Provide high spatial and temporal resolution. This may require measure-ments over submicrometre distances and in the millisecond range.

(d) Not interfere with normal physiological activities or cellular responses(i.e. be as non-invasive as possible).

3. Imaging intracellular free calcium and pHIon imaging is a technique which typically combines microscopy with the useof ion-sensitive fluorescent probes in order to study intracellular ion concen-trations. Imaging ion-sensitive fluorescent dyes in living cells can be used tofulfil all of the criteria defined in the previous section to establish a causalrelationship between a change in Ca2+ (or pH) and a cellular response to aspecific stimulus. (For information on other methods of ion measurementconsult refs 12-16.) The key aspects of intracellular ion imaging with fluores-cent dyes are:

(a) The use of Ca2+- and pH-sensitive dyes which are introduced into cells(Sections 4 and 5). These ion reporters allow intracellular ion concentra-tion to be determined optically by virtue of their emission of photons(fluorescence) in a manner which is dependent upon ion concentration.

(b) The use of microscope optics (Section 6) to focus the optical signal fromthe intracellular ion-sensitive probes onto a suitable detector (Section 7)to form an image.

(c) The quantitative relationship between ion concentration and photonsemitted by ion-sensitive dyes which allows quantitative determination ofion concentration (Sections 4 and 12). The exact quantitative relationshipis dependent upon the properties of the probe (Section 4.3).

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6: Calcium and pH imaging in living cells

Ion imaging can provide quantitative, spatially and temporally resolvedinformation on intracellular Ca2+ or H+ concentrations as they undergodynamic changes within the living cell. In many cases, imaging may also beconsidered less invasive than other methods of intracellular ion measurementand so able to give a better picture of normal signal-response coupling. Thetechnique lends itself well to being used in conjunction with the experimentalmanipulation of living cells and can even be employed simultaneously withother methods of ion measurement (e.g. ion-sensitive microelectrode tech-niques; see Section 14). Ion imaging, however, is not without its problems andlimitations, which are discussed throughout this chapter.

4. Fluorescent dyes for free calcium and pH4.1 Properties of calcium and pH dyesIon-sensitive dyes are fluorescent molecules (i.e. they absorb light at onewavelength and emit light at a longer wavelength) which reversibly bind tospecific ions. A measure of the affinity of ion binding to the dye is the dissoci-ation constant (Kd) for that dye-ion interaction. Binding of the ion causesconformational changes in the dye molecule altering its fluorescence excita-tion and emission properties which can be used to report ion concentration(Figure 1).

Many different fluorescent dyes are commercially available for measuringCa2+ concentration and pH (see Tables 1 and 2) (17), and it is important toselect the most appropriate dye for one's needs. Important parameters toconsider are:

(a) Dissociation constant (Kd for Ca2+ dyes or pKa for pH dyes). Determinesthe range over which ion concentrations can be measured, generally 0.1 XKd to 10 X Kd (see Tables 1 and 2), but this range can be limited by otherfactors (e.g. dye precipitation, dye brightness). In the case of dextran con-jugated dyes the actual Kd will vary between batches.

(b) Ion selectivity. Determines how strongly the dye binds the ion of interestrelative to other ions (e.g. Mg2+, H+, and K+) likely to be present in vivo.

(c) Spectral properties. Dictate the imaging procedures which may be used(see Figure 1, Tables 1 and 2, Sections 4.3 and 6.3) (17).

(d) Kinetics of photon emission and ion-dye interaction. Determines howwell rapid, transient changes in ion concentration are reported (18).

(e) Photon absorption and quantum yield. Determine efficiency with whichdye absorbs and emits photons. These dictate the effective dye brightnessand hence the concentration of dye required (17).

(f) Photobleaching. Results in irreversible destruction of the excited dye (17).

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Figure 1. Excitation and emission spectra of selected Ca2+ and pH dyes determined inaqueous buffer solutions in vitro by Molecular Probes. (A) Oregon Green 488 BAPTA-1,a single wavelength dye. (B) lndo-1, a dual emission ratiometric dye. (C) Fura-2 10 kDadextran, a dual excitation ratiometric dye. (D) Fluo-3/Fura Red, a dual excitation ratiometricdye-pair. (E) Calcium Green-1/Texas Red 70 kDa dextran, a dual excitation-dual emissionratiometric dye-pair. (F) BCECF, a dual excitation ratiometric dye. (G) Carboxy SNARF-1, adual emission ratiometric dye. Reproduced from ref. 17 with permission.

224


This can significantly reduce the fluorescence signal and distort therelationship between ion concentration and fluorescence during an experi-ment (19).

(g) Cytotoxicity. Determines the dye concentration tolerated by the speci-men and varies considerably between different dyes, loading methods,irradiation conditions, and different cell types.

(h) Size, charge, and hydrophobicity. Influence the intracellular behaviourand loading properties of dyes (Sections 4.2 and 5).

4.2 Intracellular dye behaviourIt is now well established that dye-ion interaction and dye spectral responsesare significantly influenced by various factors within the intracellular micro-environment, including the concentration of other ions, hydrophobicity,viscosity, and temperature (20-22). The interpretation of dye imaging resultsis complicated because the precise nature of the intracellular microenviron-ment influencing intracellular dye behaviour is unknown (Section 12.5).Indeed, intracellular dye behaviour is arguably the biggest problem in ionimaging. The influence of intracellular pH on Ca2+ dyes is a particularly

Figure 2. cSNARF-1 pH response examined in vitro. Dye solutions (50 uM) were ratioimaged using a laser scanning confocal microscope (Bio-Rad MRC600). Ratio values (580 nm/640 nm) at defined pH were compared for MES/Hepes buffer solutions and an artificialcytosol preparation comprising: 25% (v/v) ethanol, 60% (w/v) sucrose, 10 mM MES, 10 mMHepes, 100 mM KCI, 20 mM NaCI, and 1 mM MgSO, (24). Fluorescence intensities at bothwavelengths were roughly twice as high in artificial cytoplasm as in buffer alone. Fromref. 30 with permission.

225

Table 1.Ca2+

Ca2+ dye

Bis-furaBTCCalciumCrimsonCalciumGreen-1CalciumGreen-2CalciumGreen-5NCalciumGreen-C18CalciumOrangeCalciumOrange 5NCalciumGreen-1Texas RedFluo-3

Fura-2Fura-C18

Fura-Indoline-C18

•sensitive fluorescent dyesa

TypicalexcitationX (nm)b

340/380400/480570

490

490

490

490

545

545

488/568

490

340/380340/380

500/610

TypicalemissionX (nm)6

510540615

530

535

530

530

575

580

535/615

525

510510

700

FreeCa2+

range(uM)c

0.04-40.7-70.02-2

0.02-2

0.06-6

1.4-140

0.03-3

0.02-2

2-200

0.04-4

0.04-4

0.02-20.02-2

0.04-4

AM esterd Dextranfluorescent conjugate

(Mr, kDa)

NAe

+ NA+ 10,70

3, 10, 70, 500

NA

NA

NA NA

+ NA

+ NA

NA 70

NA

+ 3, 10, 70NA NA

NA NA

Notes

Higher fluorescence output than Fura-2

More photostable thanCalcium Green-1 or Fluo-3More fluorescent thanFluo-3 in Ca2+ free and Ca2+ bound formsLarger fluorescence increase upon bindingCa2+ than Calcium Green-1

Lipophilic derivative for detecting Ca2+ nearmembrane surfacesMore photostable than Calcium Green-1 or Fluo-3

More photostable than Calcium Green-5N

This dye consists of Ca2+-sensitive CalciumGreen-1 and Ca2+-insensitive Texas Red linkedto 70 kDa dextranMagnitude of Ca2+-dependent fluorescenceincrease greater than for Calcium Green-1More resistant to photobleaching than lndo-1Lipophilic derivative for detecting Ca2+ nearmembrane surfacesLipophilic derivative for detecting Ca2+ nearmembrane surfaces

Fura Red

lndo-1Mag-Fura-2Mag-Fura-5Mag-indo-1MagnesiumGreenOregonGreen 488BAPTA-1OregonGreen 488BAPTA-2OregonGreen 488BAPTA-5N

490420/480350340/380340/380350490

490

490

490

660>550405/485510510405/485530

520

520

520

0.02-2

0.03-32.5-2502.8-2803.5-3500.6-60

0.02-2

0.06-6

2-200

Quin-2Rhod-2

340540

495570

0.01-10.06-6

NA

10, 70NANA10, 70NA

10, 70

NA

NA

NANA

Fluorescence decreases on binding to Ca2+;fluorescence weaker than with other Ca2+ dyes

More efficiently excited than Fluo-3 or Calcium

Green-1More efficiently excitedthan Calcium Green-2

Magnitude of Ca2+-dependent increasegreater than OregonGreen 488 BAPTA-1First generation Ca2+ dye

* Adapted from information in Haugland (17). All dyes shown are available from Molecular Probes. All dyes are available in free acid and esterified forms exceptCalcium Green-Texas Red.b Other wavelengths close to the values quoted will usually produce satisfactory results. Using different wavelengths may be desirable in some cases to mini-mize problems of autofluorescence, or cross-talk between channels when more than one dye is used and where emission spectra of the dyes overlap (Sections4.3, 6.4, and 12.4).c The approximate ranges over which free Ca2+ concentration can be measured using each dye have been calculated from their Kd values (Section 4.1), takinginto account that Ka values are usually higher in vivo than in vitro. Kd values used in this table were those obtained in vitro by Molecular Probes and are given inHaugland (17). Note that Kd values vary between different batches of dextran-conjugated dyes.d Fluorescence of the AM ester can be a major source of error in Ca2+ measurement.' NA = not available.

+

++++

++

Table 2. pH-sensitive

pHdye

ACMACarboxySNAFL-1

CarboxySNAFL-2

SNAFL-calcein

HPTS (pyranine)

CarboxySNARF-1ChloromethylSNARF-1LysoSensor BlueDND-192LysoSensor GreenDND-153SNARF-calceinBCECFFluorescein

CarboxyfluoresceinCarboxydichloro-fluoresceind

Carboxydimethylfluorescein

fluorescent dyesa

TypicalexcitationX(nm)b

410505/540514505/540514505/540514450/405(470/380dextran dye)488 or 514488 or 514

375

440

488 or 514490/450490/450

490/450490

490/450

TypicalemissionX(nm)b

470600540/630620540/630600540/630550530

580/640580/640

425

505

580/640530520

520530

520

UsefulpHrange

7.6-9.67.2-8.2

7.2-8.2

7.2-8.2

7-8

6.5-86.5-8

6.5-8

6.5-8

6.5-86.5-7.56-7.2

6-7.26-7.2

6-7.2

Dextranconjugate(Mr, kDa)c

NA10, 70

NA

NA

NA

NANA

NA

NA

NA10, 40, 703, 10, 40, 70,5-40 millionNANA

NA

Notes

Apparently binds to membranes

Better retained within cells thancarboxy SNAFL dyesLow cost; no membranepermeant form of HPTSavailable; caged form of dye free acid available

Better retained with cells thancarboxy SNARF-1Fluorescence increases onbinding H+

Fluorescence increases onbinding H+

Better retained with cells than carboxy SNARF-1Has been most widely used pH dyeRapidly leaks out of cells; cagedform of dye free acid availableBetter retained within cells than fluorescein

Better retained within cells than fluorescein

Cell tracker greenCMFDAFluorosenicsulfonic acidLysoSensor BlueDND-167LysoSensor GreenDND-189Oregon Green 488carboxylic acidOregon Green 500carboxylic acidOregon Green 514carboxylic acidCI-NERFDM-NERFRhodol Greencarboxylic acidLysoSensor Yellow/Blue DND-160

490/450

490/450

375

445

490/440

500

510/450

510/450510/450490

520

520

425

505

520

525

530

540540520

6-7.2

6-7.2

4.5-6

4.5-6

4.2-5.7

4.2-5.7

4.2-5.7

4-64-64-6

NA

NA

NA

NA

10,70

NA

10,70

10, 7010, 70NA

360 540/440 3-5 NA



Fluorescence increases on binding H+

Fluorescence increases on binding H+

More photostable than fluoresceinMore photostable than fluoresceinMore photostable than fluorescein

' Adapted from information in Haugland (17). All dyes shown are available from Molecular Probes. All dyes are available in free acid and cell permeant forms,with the exception of HPTS.b Common excitation and emission wavelengths for both free and dextran-conjugated dyes (except where stated). Dyes with two excitation or two emissionwavelengths presented in the form x/y are ratiometric dyes. (See footnote b in Table 1.)c NA = not available.d A range of polyfluorinated fluoresceins are also available (17).

Richard M. Parian and Nick D. Read

serious problem (23). The effects of different environmental conditions upondye response can be examined in vitro (Figure 2) (24).

Dye retention within cells and sequestration within subcellular organelles(Figure 3) is also included amongst the problems of intracellular dyebehaviour and depends upon the dye, the cell type, and the method of loading(Section 5). This, together with toxic dye effects and photobleaching, islargely responsible for defining the 'time window' over which useful data fromdye imaging may be collected. Recently the use of pressure injected dextranconjugates of dyes (Section 5.7) has improved dye retention and reducedsequestration (17, 25-30), although the fluorescence of dextran dyes is stillinfluenced strongly by the intracellular environment (27).

4.3 Single wavelength dyes, ratiometric dyes, andratiometric dye-pairs

Two major classes of ion-sensitive dyes exist: single wavelength dyes and dualwavelength (ratiometric or ratio) dyes (17, 31, 32). With single wavelengthdyes, the intensity of the fluorescence emission spectrum increases in propor-tion to the free ion concentration (Figure 1A). The problem with using asingle wavelength dye is that it is difficult to distinguish between differences inion concentration and variations in dye brightness caused by factors such asthe dye concentration, dye photobleaching, and dye leakage from a cell (seeSection 7.2). This makes absolute measurement of ion concentration withsingle wavelength dyes difficult (33). Ratiometric dyes provide a solution tothese problems because they exhibit a spectral shift upon binding to the ion ofinterest (31, 32) (Figures IB, 1C, 1E-G). In ratio imaging, two fluorescenceimages of a cell loaded with a ratiometric dye are detected at appropriatewavelengths and the ratio of fluorescence intensities for the image pair calcu-lated (Sections 11 and 12). In principle, this ratio is independent of theamount of dye measured and proportional to the free ion concentration,allowing for improved calibration of ion concentration There are three typesof ratiometric dyes:

(a) Dual excitation ratiometric dyes. These dyes are usually excited sequen-tially at two wavelengths and two images are sequentially collected at asingle emission wavelength. With Fura-2, the two excitation wavelengthsspecifically excite the ion binding form of the dye at the shorter wave-length and the ion free form of the dye at the longer wavelength. As thefree Ca2+ concentration increases there is a shift in the excitation to shorterwavelengths (Figure 1C). With BCECF, the excitation wavelengthscommonly used correspond to the pH-sensitive and the pH-insensitive(isosbestic point) parts of the emission spectrum (Figure IF).

(b) Dual emission ratiometric dyes. These dyes are excited at one wavelengthand fluorescence emissions detected at two longer wavelengths. WithIndo-1 and cSNARF-1, the two emission wavelengths correspond to the

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ion binding and free forms of each dye. As the concentration of free Ca2+

or protons increases there is a shift in the emission spectra of Indo-1 orcSNARF-1, respectively, to the shorter wavelengths of the ion bindingforms of these dyes (Figures 1B and 1G).

(c) Dual excitation-dual emission ratiometric dyes. These dyes are excitedsimultaneously or successively at two wavelengths and their fluorescencedetected (again simultaneously or successively) at two longer wave-lengths. An example of such a dye is SNAFL-calcein for pH (34).

Ratio imaging can also be performed with a 'ratiometric dye-pair' (34, 35)composed of two fluorochromes. Different dye-pair combinations are cur-rently used for Ca2+ imaging:(a) Two different Ca2+-sensitive dyes, e.g. Fluo-3 and Fura Red, (Figure 1D)

which provide a combined emission spectrum that exhibits an overall shiftto shorter wavelengths as the free Ca2+ concentration increases.

(b) Ca2+-sensitive dye and a Ca2+-insensitive 'volume marker' dye, e.g. CalciumGreen-Texas Red (Figure 1E), as a single dextran conjugate or asindependent dyes; Fluo-3 and cSNARF-1 imaged at its pH-insensitiveisosbestic wavelength (Figure 1G).

The ratiometric dye-pair strategy is adopted because of the lack of Ca2+-sensitive ratiometric dyes which can be excited at visible wavelengths (seeTable 1). Ratiometric dye-pairs are not used for pH imaging because of thewide availability of ratiometric pH dyes excited with visible light (Table 2).

5. Introducing calcium and pH dyes into living cells5.1 General considerationsThe introduction of dye into cells is fundamental to dye-based imaging tech-niques and is often the major stumbling block that decides whether or not thetechnique can be applied to a particular organism or cell type. The importantpoints to consider when selecting a loading method are given below. The firstfour points may be taken as the basis for assessment of the success of anyloading procedure:(a) Introduction of sufficient dye into cells and the degree of control over the

amount of dye introduced.(b) Dye localization within the cell compartment of interest (e.g. the cytosol

or specific organelles).(c) Dye retention within the cell compartment of interest.(d) The level of perturbation of cells by the dye itself and the loading procedure.(e) The number of cells that can be loaded with dye in a given time.(f) The relative ease of different loading procedures and the type of equip-

ment required.Loading cells with dyes is not an exact science and generally involves some

231


compromising of the above requirements. So far there is no single loadingmethod which can be successfully applied to all cells and no absolute way ofperforming any loading method. Dye loading methods can be divided intotwo basic types:

(a) Methods based on cell permeant dye forms (e.g. ester loading and low pHloading).

(b) Methods based on cell impermeant dye forms (e.g. electroporation andmicroinjection).

In general, methods involving permeant dye forms are easier to performbut pose more of a problem with dye sequestration within organelles. Methodswhich involve cell impermeant dye forms are more difficult but often affordbetter control over dye delivery. The most commonly applied methods arediscussed in Sections 5.1 to 5.7, and all have their relative merits and draw-backs. Making use of loading techniques and conditions described by otherresearchers can make life easier and at least one general reference for eachmethod has been given below. However, small differences in the loading con-ditions, age and treatment of the dye, the state of the cells or tissue used, andthe specific cell type, can critically affect loading success. Generally, the bestloading method and conditions must be determined empirically for one's par-ticular organism and cell type. Protocol 1 is a description of ester loading, themost commonly used dye loading method, although many of the points raisedcan be applied to the other methods.

5.2 Ester loadingEster loading (37) generally employs the cell permeant acetoxymethyl (AM)esterified form of dyes (Protocol 1). The ion-insensitive, esterified form of thedye is lipophilic allowing it to cross membranes. Once in the cytoplasm the estergroups are cleaved off by endogenous esterases releasing the ion-sensitivefree acid form of the dye. Whilst the technique has been applied to most celltypes of animals, plants, and fungi, success is very dye-, cell-, and condition-specific. For instance AM esters of Ca2+ dyes load poorly or not at all intowalled plant and fungal cells (33) whilst the AM esters of many pH-sensitivedyes are rapidly taken up by these cell types and cleaved to release intracellularfree dye. Animal cells are generally easier to ester load with Ca2+ dyes. Severalproblems with the ester loading method have been recognized (21, 38):(a) Incomplete ester hydrolysis within the cytoplasm leading to the loading of

organelles.(b) Active uptake of dye free acid from the cytosol into organelles (39)

(Figure 3).(c) Poor retention within cells of the dye released by ester hydrolysis.(d) Cytotoxicity of AM esters and/or of the formaldehyde and acetic acid

released within cells by ester hydrolysis.

232


(e) Altered intracellular spectral properties of ester loaded dye because ofincomplete cleavage of the ester groups.

(f) Background fluorescence of uncleaved dye ester (with some dyes only;see Table 1) or extracellular free dye produced by the action of extra-cellular esterases.

Various ways have been devised to improve ester loading:

(a) Using a non-cytotoxic detergent such as Pluronic-F127 (Molecular Probes,Inc.) to disperse dye ester evenly.

(b) Loading at low temperature to reduce sequestration within organelles (21).

(c) Continuous loading throughout an experiment (30).(d) ATP-induced permeabilization of membranes in some cell types (40).(e) Using dye forms such as cSNARF-calcein (17) and Fura-PEl (39) which

can be ester loaded but have improved properties of localization andretention within the cytosol.

Protocol 1. Loading cells with the fluorescent dye cSNARF-1 bythe ester methoda

2. Prepare cells. In some cases it is desirable to hold cells down and asimple method is to use polylysine coated coverslips (polylysineapplied to coverslips as a 1:100 dilution of the 1% (w/v) stock solutionfor 30 sec, then washed off for 5 min in running water) (see Section 9).

3. Apply the dye solution (or culture medium containing dye solution) tothe culture chamber to give a final dye concentration of 2-20 uM.

233

Equipment and reagents

Either an epifluorescence microscope withlow light camera or a confocal microscope,equipped with a suitable filter set for imag-ing cSNARF-1 (excitation at 488 nm, emis-sion > 540 nm). Alternatively, for ratioimaging the following filter set may beused: excitation 514 nm; emission-1 580 ±15 nm; emission-2 640 ± 20 nm.Ultrapure HPLC water

A culture chamber allowing access to cells(41)cSNARF-1 AM ester (Molecular ProbesInc.): 20 mM stock in DMSO (dimethyl sul-foxide, Sigma) stored at -70°CPluronic F-127 (Molecular Probes Inc.): 2%(w/v) aqueous stock stored at -10°CAppropriate cell culture media

Method

1. Prepare the dye solution by mixing a 2 ul aliquot of 20 mM stockcSNARF-1 AM ester in DMSO and 198 ul of Pluronic F-127 (0.04%, w/v)made up with ultrapure HPLC water. The concentration of the solventDMSO is kept as low as possible, usually < 1% (v/v). Generally, aqueousdye ester solutions are best made up fresh and used immediately.However, the 200 uM cSNARF-1 AM stock can last for a week if kept at-10°C.



4. Observe uptake of dye into cells by fluorescence microscopy. Simpleexamination of total fluorescence emission is of initial value but it isbetter to carry out an analysis of dye uptake using the same imagingsystem which will be later used for ion imaging. Examination of fluor-escence at each of the two cSNARF-1 emission wavelengths allowsloading conditions to be optimized for subsequent ratioing. It is import-ant to make observations of the time taken for dye uptake into cells,the location of dye within cells, and any changes in dye location withincreasing loading time. It is also important to examine cellular auto-fluorescence at the imaging wavelengths prior to assessment of load-ing (Section 6.4).

5. Where required, terminate loading by dilution with fresh medium, orby washing out excess dye by perfusion or exchange of medium in thechamber, (However, continuous loading with a low dye concentrationmay sometimes be found to be the best method.)

6. Assess variation in loading within the cell population (Section 5.8).7. Assess the viability of individual dye loaded cells (see Section 15).

•'Note that this protocol, with the exception of the precise equipment set-up and dye used,would be generaIly appropriate for ester loading all Ca21 and H1 dyes.

Figure 3, Confocal images of cSNARF-1 ester loaded into a growing Dryopteris affinisrhizoid. (A) 10 min after a 15 min loading period with 3 uM cSNARF-1 AM (see Protocol 1),Vacuoles excluding dye appear dark. (B) The redistribution of dye 45 min after ester load-ing. The vacuoles and cytoplasm are hard to distinguish from each other because dye ispresent in both. (C) Three hours after ester loading most of the dye has been accumu-lated within the vacuolar system. Bar = 15 um. From ref. 30 with permission.

234


5.3 Low pH loadingIn low pH loading (42, 43) the pH of the loading medium is lowered to a value(typically between pH 3-5) at which the acid groups of the free dye becomeprotonated (and hence uncharged) and the dye is able to directly cross theplasma membrane. Within the cytosol the pH is higher, the dye becomes dis-sociated, and the charged molecules are trapped in the cell. The method ismost commonly used with walled plant cells. However, certain potentialdrawbacks of this method are:

(a) Loading is usually very slow and the amount of dye loaded is often lowcompared with other methods.

(b) Cells must be tolerant of a low external pH.

(c) Sequestration of free dye within organelles is common.

(d) Washout of excess extracellular dye before imaging is critical.

(e) Dye often becomes irreversibly bound to the walls of plant or fungal cells.

5.4 Scrape loadingScrape loading (44) is applied to adherent cell cultures and involves tran-siently permeabilizing cell membranes by physical perturbation to allowmembrane impermeant dye free acid or dye dextran conjugates to enter cells.The dye is trapped once membrane integrity has recovered. The mainproblems of the method are:

(a) Long recovery times required.

(b) Loss or sequestration of dye free acid (avoided when using dextran con-jugates).

(c) The need to remove excess dye from around cells before imaging.

5.5 ElectroporationElectroporation (45, 46) involves applying short electrical pulses to cellswhich, by temporarily permeabilizing cell membranes, can allow dyes to leakin. It is very important to optimize electroporation conditions for the cell typeused. Variables which need to be controlled include: concentration of dye inthe surrounding medium, distance between the electrode and cells, voltageapplied, number of electrical pulses, pulse frequency, capacitance, and thecomposition of the 'poration' medium. Careful attention should be paid to thecomposition of the 'poration' medium because of its influence over the electri-cal treatment. Standard culture media will often have to be modified (46). Themethod has been mostly used with animal cells (46), other non-walled cellssuch as plant cell protoplasts (33), and also some walled cells such as pollengrains and yeast cells (47, 48), Cell suspensions are most commonly used,

235


although the technique has been extended to adherent cells (46). The mainproblems of the method are:

(a) Electroporation equipment required.(b) The potential for cell damage which necessitates caution in its application

(45). Checks on cell viability are critical (e.g. by assessing membraneintegrity with a membrane impermeant dye such as propidium iodide).There is evidence that cells are able to reseal their membranes within fiveseconds if treated carefully (46).

5.6 lonophoretic microinjectionlonophoretic microinjection (49, 50) is the easier to perform of the twomicroinjection methods. The cell is impaled with a glass micropipette and thenegatively charged free acid of the dye driven in directly by applying a nega-tive current. Some cell types are better suited than others for microinjection.Large, round cells (e.g. oocytes) are the easiest to inject whilst small, turgid,and vacuolate walled cells (e.g. plant guard cells) are the most difficult. Inmany cases microinjection is the only way to load plant and fungal cells effect-ively. Problems with the method are:

(a) Microinjection requires substantial apparatus (micromanipulators, amicropipette puller, a suitable power supply, needle holder, and capillaryglass for making micropipettes). With more difficult cell types (e.g. walledplant and fungal cells), microinjection can be a very laborious and pains-taking procedure requiring a high degree of dexterity and patience.

(b) One of the greatest difficulties with microinjection is to obtain the 'ideal'injection needle and then to generate multiple needles with the samecharacteristics. This is crucial with cells which are difficult to inject. Pro-grammable, multistage pipette pullers are useful to maintain reproduc-ibility (e.g. the Sutler P-97 micropipette puller; Sutler Instrument Co.).

(c) Damage to cells caused by impalement or, more often, when removingthe needle.

(d) The ability of many cells to expel or sequester dye free acid after it isintroduced into the cytosol (50).

(e) Cells may be perturbed by the applied current.(f) Only cells larger than 5-10 um in diameter can be injected.(g) Only charged molecules up to — 10 kDa can be injected.

5.7 Pressure microinjectionUnlike ionophoretic injection, pressure injection involves forcing a volume ofdye into cells by applying pressure (38, 51, 52) (see Chapter 10). Most of thepoints covered in the previous section on ionophoretic microinjection also

236


apply here. Pressure injection is commonly more difficult to perform thanionophoresis, although it is usually much easier to use in animal cells (wherethe technique can be automated) than plant or fungal cells. The micropipettesused for pressure injection tend to have wider apical apertures than for iono-phoresis, especially for injecting the more viscous dextran-conjugated dyes,and this can be more damaging to cells. An important advantage over iono-phoresis is that, in principle, pressure injection allows the delivery of virtuallyany size of charged or uncharged molecule into cells. It is particularly useful forintroducing ratiometric dye-pairs (Section 4.3) at specific relative concentrations.

Several different pressure injections systems exist and some use compressedgases whilst others involve liquid compression. Crude systems have little finecontrol over the pressure applied whilst more complex systems, such as thoseemploying a pressure probe (51), provide very precise regulation of the applica-tion of pressure, and a digital readout of the pressures involved. An advantageof the pressure probe for use with walled, turgid plant, and fungal cells is thatthe system can be pressurized before injection, with the needle held against thecell wall, to prevent cell turgor pressure forcing cell contents into the injectionneedle upon impalement. Although in many ways pressure microinjectionwould appear to be the desired method for introducing dyes into cells, the tech-nical difficulties involved and the small numbers of cells which can be loadedare often significant justification for the use of the other methods.

5.8 Quantifying the extent of dye loadingThe concentration of intracellular dye is important because it determines thebrightness of the fluorescence signal from the cell. However, dye toxicity andpossible intracellular buffering effects of the dye on the ion of interest, alsoneed to be considered. A means of assessing the extent of loading, at least inrelative terms, is important, especially as there is usually significant cell-to-cellvariation in the amount of dye loaded. Absolute quantification on the basis offluorescence is difficult because of differences in dye brightness in vivo and invitro even when the effect of ion concentration has been taken into account(21). Nevertheless, comparing the relative fluorescence brightness of cellsloaded with dye under defined imaging conditions is a useful measure. Withpressure microinjection intracellular dye concentration can be estimated fromthe injected volume (20).

6. Equipment for fluorescence microscopy6.1 Fluorescence microscopesThe main microscope manufacturers provide a range of fluorescence micro-scopes suitable for ion imaging. The points to look for in a microscope are:

(a) An epifluorescence microscope with appropriate dye excitation source(Section 6.3).

237

Richard M, Parton and Nick D. Read

(b) Transmission optics which allow correlative microscopy with bright-field,phase-contrast, and/or differential interference contrast optics.

(c) A filter arrangement which allows the rapid (0.1 sec or faster) selectionof different excitation and/or emission wavelengths, and which shouldideally be automated.

(d) Suitable ports for the attachment of one or more image detectors (typi-cally low light cameras).

(e) A specimen stage providing good access to the specimen to allow formicromanipulation and experimental treatments.

Two main types of microscope are used: upright microscopes, in which theobjective is above the stage; and inverted microscopes, in which the objectiveis located below the stage. For the majority of ion imaging applications, theinverted configuration is preferable because it:

(a) Allows the use of high magnification, high numerical aperture, shortworking distance objectives, in conjunction with slide culture/perfusionchambers.

(b) Provides good access for microinjection.(c) Avoids the requirement for placing a coverslip on top of the sample

which can result in living cells being deprived of oxygen during the courseof an experiment.

Upright microscopes are particularly useful when cells on the surface of alarge opaque specimen (e.g. a leaf) needs to be microinjected but this requiresthe use of long working distance lenses.

6.2 ObjectivesImportant considerations when selecting an objective for fluorescence workare:

(a) Numerical aperture (NA): important for the efficiency of light collectionand in determining the axial and lateral optical resolution.

(b) UV transmission: important for use with UV excited Ca2+ dyes and forphotoactivation of caged probes (Section 14). Many high NA plan apoobjectives do not transmit light at wavelengths below 360 nm sufficientlywell for these purposes.

(c) Correction for axial chromatic aberration (particularly important for con-focal microscopy). Specialized lenses corrected for chromatic aberrationat UV wavelengths are necessary for UV confocal microscopy.

(d) Working distance and coverslip thickness correction. The most appropriateobjective to use will vary depending upon whether an inverted or uprightmicroscope is used and whether single cells or thick tissues are examined.

For further discussion about objectives refer to Chapters 1 and 2.

238


6.3 Dye excitation sources for ion imagingThe main illumination sources for exciting fluorescent dyes in ion imaging are:

(a) Mercury arc lamp. Has an uneven spectral output with emission highenough for dye excitation only at specific wavelengths: 313, 334, 365, 405,436, 546, and 577 nm. Note that these wavelengths are suboptimal fordual excitation ratioing of the important Ca2+ dye, Fura-2.

(b) Xenon arc lamp. Provides a much more even emission over the 300-700nm range than mercury lamps and is, therefore, more suitable for imagingwork requiring dye excitation at both UV and visible wavelengths. Aproblem of xenon lamps is the high emission in the IR which can lead toheating of the specimen unless a heat filter is used in the optical path.

(c) Lasers. Used as an excitation illumination source primarily in confocaland multiphoton imaging (Chapter 2). Although lasers only provide exci-tation at discrete wavelengths, a range is available which covers most ofthe useful visible and UV wavelengths for ion imaging. The most commonlasers used for Ca2+ and pH imaging are: krypton-argon (emission at 488,568, and 647 nm); argon (emission at 488, 514, and sometimes 457 nm);UV-argon (emission at 351, 364, and sometimes 457, 488, and 514 nm);and helium-cadmium (emission at 442 nm). There is an increasing trendfor confocal microscopes to be fitted with multiple lasers to compensatefor the limited lines available from individual lasers.

6.4 Filters for ion imagingA filter set comprises one or more excitation filters, emission filters, anddichroic mirrors (Figure 4) arranged to isolate the most appropriate excitationwavelength(s) and collect the required fluorescence emission wavelength(s).The three main types of filter used are:

(a) Band width (band pass or barrier) filters. A variety of abbreviations areused for these filters including DF (discriminating filter), BP (band pass),NB (narrow band), and WB (wide band). They are characterized by onlytransmitting light within a defined spectral range and are used as bothexcitation and emission filters.

(b) Long pass (LP) filters transmit all wavelengths above the stated value.(c) Dichroic mirrors or beam splitters (DRLP) reflect light of shorter wave-

lengths than the stated value and transmit all wavelengths longer thanthat value. They are used to separate excitation light from the higherwavelength spectrum of the fluorescence signal, or for separating differ-ent parts of the emission spectrum to two detector channels (e.g. for emis-sion ratio imaging).

Complete filter sets are commercially available for most Ca2+ and pH dyesfrom microscope manufacturers and Molecular Probes. Individual filters can

239


be purchased to assemble a customized set for particular needs from variousmanufacturers including Omega Optical, Ditric Optics, Baling Electro-Optics,Glen Spectra, and Oriel. When assembling a customized filter set from indi-vidual filters it is useful to start by examining the excitation and emissionspectra of the dye (17) (Figure 1). It is particularly important to maximize thecollection of useful dye signal whilst discriminating against contaminatingcellular autofluorescence or background fluorescence. It is also important tocheck the literature for information on dye spectral changes which have beenreported to occur within the intracellular environment (Section 4.2). Forexample, imaging Ca2+ with Fura-2 is significantly influenced by the nature ofthe intracellular environment when examined with 340/380 nm excitationratioing (emission 510 nm). However, these problems may be reduced by340/365 nm excitation ratioing (20).

7. Fluorescence imaging systems7.1 General requirementsThe main components of the fluorescence equipment required for imagingion-sensitive dyes are as follows (Figure 4):

(a) Fluorescence microscope set-up as described in Section 6.(b) Fluorescence detector (video camera or photomultiplier tube).(c) Computer hardware and software for image capture, processing, analysis,

and storage.(d) Hardware for producing hard copies of images (e.g. a slide maker or suit-

able printer; see Section 13.2).

Selecting the most appropriate imaging equipment depends on one'sspecific imaging applications and budget. Of critical importance for ion imagingis the image detector used (53-55). Features of the image detector which needto be considered are:

(a) Quantum efficiency. The number of the photons arriving at the detectorwhich are actually detected (i.e. recorded as a voltage readout).

(b) Sensitivity and noise. Sensitivity is related both to the quantum efficiencyof the detector and the degree of noise which it generates. It may bethought of as the lowest level of photons which can be detected withan acceptable signal-to-noise ratio (Rs/n) (see Section 12.2 for furtherdiscussion).

(c) Linearity. The relationship between input signal (photons) and outputsignal (voltage).

(d) Spatial resolution. The extent to which this is limited by the detectorusually depends on the number of scan lines in video-based systems or the

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Figure 4. Conventional fluorescence imaging system allowing simultaneous bright-field(transmitted light) and epifluorescence imaging. BF-IS bright-field illumination source,TF-620LP = transmitted light imaging filter, DIC-0 = DIC optics, C condenser, SC =specimen chamber, MS = microscope stage, OBJ = objective, P = Wollaston prism, EX-IS = dye excitation light source, DM - dichroic mirror, EX-FC = excitation fitter changer,F = filter, EM-FC = emission filter changer, AN analyser for DIC, VS = video signal,VDU = video display unit. * Each camera requires a frame grabber card; for somecameras a specific, compatible type of card is required.

size and arrangement of CCD detector elements (which relates to pixelsin the image).

(e) Temporal resolution. This is limited by the rates of:(i) image detection,(ii) readout from the detector,(iii) digiti/ation lime for the video signal from the camera.

7.2 Conventional fluorescence imagingIon imaging commonly combines the use of a conventional fluorescencemicroscope with a low light camera detector. The main disadvantage of thisapproach is that there is little discrimination between fluorescence fromwithin the focal plane and 'out-of-focus' (including stray) fluorescence which

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originates from above and below it (Figure 5). The inclusion of iiul-of-focusinformation and the detection of the average fluorescence over the full widthof the focal plane limit spatial resolution and image quality (see Section 4.3).Confocal imaging (Section 7.3) or multipholon imaging (Section 7.4) over-come these limitations (see Chapter 2). However, attempts to reduce theseproblems by deconvolution procedures (56) are not to be recommended forquantitative ion imaging because of the way in which the mathematical trans-formations used alter numerical data within images.

With imaging systems employing low light camera detectors, light from thewhole imaged field is detected simultaneously, although the data may besubsequently read off the detector serially. This can allow very rapid imagedetection or averaging of several successive frames to produce less noisyimages. Images may be captured on video or, more commonly, digitally in acomputer framcstore. Camera-based systems are of two main types: tubecameras and CCD (charged coupled device) cameras (Chapter 3) (53, 54).Currently the best camera for most ion imaging applications is a cooled CCD.Recent examples of such cameras are able to collect images at close to videorate (e.g. 18.9 frames per second for a 512 X 512 pixel image size digitizedto 12-bits per pixel using the AstroCam Model UltraPix FE250; AstroCam,personal communication).

Figure 5. Comparison of dye excitation, photobleaching, and collection of fluorescenceemissions at the specimen in conventional, confocal, and multiphoton imaging (seeSection 7). The region of excitation shown for conventional microscopy is with the excita-tion iris closed down to confine irradiation to only the cefl of interest. For confocal andmultiphoton imaging only a single scanned spot is shown. This spot is scanned acrossthe image to construct the final image.

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7.3 Confocal imagingConfocal imaging is a technique in which fluorescence from only a thin opticalplane within the specimen is detected. This 'optical sectioning' ability elimin-ates the out-of-focus information normally detected by conventional imagingand provides improved spatial resolution, especially in the z axis, over con-ventional fluorescence microscopy. Confocal microscopy is dealt with in detailin Pawley (57) and Chapter 2. Briefly, the optical arrangement for confocalimaging is such that only fluorescence from the specimen originating in thefocal plane of the excitation light passes to the detector (Figure 5). Lightoriginating above and below the plane of focus is eliminated by having thefluorescence signal pass through a small aperture in front of the detector.

Confocal microscopy has found wide application in quantitative ion imagingfrom single cells to tissues and intact organs (58-60). For ion imaging, a con-focal laser scanning microscope (CLSM), which encompasses 'point scanning',a pin-hole aperture, and one or more photomultiplier tubes, is usually used.

The important advantages of confocal microscopy with respect to ionimaging are:(a) Increased 3D resolution. This allows easier identification of dye seques-

tration within organelles and better interpretation of localized differencesin intracellular ion concentration. It also minimizes problems of unevendye distribution and uneven specimen thickness which can cause seriousartefacts in conventional fluorescence imaging where light is detected,and hence the recorded signal averaged, over a much greater thickness ofthe cell.

(b) The ability to image intracellular ion concentration from thin opticalsections within thick tissue samples and intact organs.

Unfortunately confocal optical sectioning comes at the cost of:

(a) The requirement for complex and expensive equipment.(b) The potentially damaging effects of irradiating living specimens with an

intense laser beam (especially significant at UV wavelengths). In confocalmicroscopy, regions of the sample above and below the focal plane, aswell as the plane of focus, are irradiated (Figure 5).

(c) The high level of fluorescence generally required to obtain reasonableimage quality. This is mainly because fluorescence is only detected from athin optical section (see Section 12.2) and additionally, the result of signalloss along the CLSM light path.

(d) The slow rate of imaging due to the low numbers of photons which maybe collected in a given time. For this reason the Rs/n of confocal systems isoften considered poor (see further discussion in Section 12.2). Accumu-lating or averaging successive images greatly improves the Rs/n by increas-ing the number of photons collected at the cost of temporal resolution.

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7.4 Multiphoton imagingMultiphoton imaging (which includes two-photon imaging) (61) is a recenttechnology which, like confocal imaging, provides improved 3D spatial reso-lution over conventional fluorescence imaging. However, in contrast to con-focal microscopy, multiphoton imaging does not rely on any special opticalarrangement to achieve this, but on the way in which the fluorescent dye isexcited. The technique is performed using a laser scanning microscope system,usually a modified CLSM. The only commercial system currently available issupplied by Bio-Rad.

In multiphoton imaging a dye molecule is excited by light at wavelengthswhich are usually > 2 X of those used for conventional fluorescence or con-focal microscopy. Excitation by such low energy, long wavelength light ispossible only at extremely high photon fluence so that multiple (usually twoor three) photons are absorbed simultaneously and their energies roughly'add up'. Thus in principle, dyes normally excited at 350 nm can be excited bysimultaneous absorbtion of two photons at approximately 700 nm or threephotons at approximately 1050 nm. Focusing the excitation laser beam intothe specimen produces a cone of excitation illumination but only at the pointof focus is there a high enough fluence of light to achieve simultaneous two(or three) photon absorption. This highly selective excitation means that allfluorescence emission must originate from the narrow region of the specimenfocus plane and hence high 3D spatial resolution is achieved (Figure 5). Thecurrently favoured excitation source for this type of work is a mode-locked Ti-Sapphire laser producing 100 fsec duration pulses with a frequency of about80 MHz over the range 700-1050 nm.

Advantages of multiphoton imaging are:

(a) Avoidance of UV radiation damage to cells because low energy excita-tion wavelengths are used and cells are only irradiated at high fluence in anarrow focal plane.

(b) Reduced dye photobleaching and phototoxic cell damage (Section 9)because dye is only excited in a small region of the specimen.

(c) Imaging at greater depths within thick samples due to deeper penetrationby the long wavelength light used.

(d) Improvement in the axial spatial resolution of caged probe photorelease(see Section 14).

Although a complete multiphoton imaging system is commercially avail-able, the technology is still under development and there are a number ofproblems and questions which remain to be answered, including:

(a) The high cost and complexity of Ti-Sapphire lasers.(b) Dye absorbtion characteristics are very different in multiphoton excita-

tion compared with that for single photon excitation (62). Two-photon

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absorption spectra have been difficult to measure and as yet cannot bepredicted. Emission spectra are, however, similar for conventional andtwo-photon excitation (62).

(c) Possible harmful effects of high intensity, long wavelength irradiation.Specimen heating, particularly due to single photon absorbance of IRirradiation by water, is of major concern. This problem is reduced byusing pulsed rather than continuous laser irradiation.

Multiphoton imaging has been successfully employed to image free Ca2+ inliving cells using Indo-1 (61, 63) and, in this respect, seems to have significantpotential as an alternative to UV laser scanning confocal microscopy.

7.5 Imaging with multiple detectorsMany imaging systems are designed with two or more detectors (either mul-tiple cameras or photomultiplier tubes) and allow simultaneous detection ofmultiple, separate signals (64). Important applications in ion imaging are:

(a) Simultaneous collection of perfectly registered bright-field (transmitted orreflected light) and fluorescence images (Figure 4). This is important forcomparative analysis of dye fluorescence (or ion concentration) and cellmorphology, especially in fast moving or growing cells where sequentiallycollected images would not necessarily correspond.

(b) Simultaneous, dual emission ratio imaging where the emission signal issplit by a dichroic mirror into two beams, each covering a different rangeof emission wavelengths. This provides the best temporal resolution andimage registration for ratio imaging (see Section 12.4). With furtherdetector channels simultaneous bright-field imaging and ratio imaging ispossible.

(c) Simultaneous Ca2+ and pH imaging. Simultaneous ratio imaging of Indo-1 (for Ca2+) and cSNARF-1 (for H+), co-loaded into cells, can be per-formed using a four channel imaging system (such as described in ref. 64)with simultaneous excitation at 350 nm (Indo-1) and 540 nm (cSNARF-1)and simultaneous image collection at 405 and 475 nm (Indo-1) and 575and 640 nm (cSNARF-1).

8. Optimizing the performance of imaging systemsThe correct set-up and use of equipment is essential for achieving good,reproducible imaging data, especially in relation to quantitative analysis. Themost important aspects of the imaging system to pay attention to are:

(a) Optical alignment of both the excitation illumination and emission light.In confocal systems alignment can be particularly critical. Refer tomanufacturer's instructions for alignment procedures.

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(b) Routine performance checks should be carried out on equipment, par-ticularly optical alignment and detector performance. A range of fluor-escence standards for checking equipment are available (Table 3 andProtocol 2). Other test samples are often available from microscopemanufacturers. Detectors may become damaged or give false readings ifthey become saturated with light. Detector sensitivity and 'dark signal'levels are good indicators of detector performance.

(c) Care of objectives. Dirty or scratched objectives reduce fluorescencesignals and can introduce optical distortions. Objectives should bechecked regularly under a dissecting microscope and occasionally given athorough cleaning with isopropanol using a new, fine, hair bristled brush(e.g. artist's sable, size 000). Objectives should be used with No. 1.5 cover-slips (0.16-0.19 mm thick). Objectives with a coverslip correction collarshould be correctly adjusted for the coverslip thickness used.

(d) Detector settings. The correct gain and black level settings should be usedto avoid detector saturation (photons arriving at the detector above thelevel registered as maximum signal output) and detector underflow (thelevel of photons reaching the detector but registering as zero signal out-put). A voltmeter may be used to monitor voltage output from some

Table 3. Fluorescence standards

Standard Supplier Properties

Focal Check MolecularProbes

PS-Speck

63 nm latexbeads

InSpeck

MolecularProbes

15 um spheres.a Combines twostains: one throughout thecentre of the bead; the other asan outer ring.175 nm (subresolution)fluorescent beads.a

BangsLaboratories

63 nm (subresolution)fluorescent beads.c

Molecular Beads available in a range ofProbes fluorescence intensities

(0, 0.5, 1, 3, 10, 30, and 100%).a

Applications

To check fluorescenceimage registration formultiple wavelengthimaging.To determine the p.s.f.''which allows estimation ofthe optical resolution andchecking of microscopealignment.To determine the p.s.f.bwhich allows estimation ofthe optical resolution andchecking of microscopealignment.Fluorescence intensityreference source for testingsystem performance.

aAvailable in a range of excitation and emission wavelengths to cover most applications.b p.s.f. = point spread function (see Section 8, Protocol 2).c Available in three forms: excitation wavelength (nm)/emission wavelength (nm) = 360/420; 555/570;660/690.

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detectors to gain an idea of the output dark current (in the absence oflight) and the magnitude of the output signal with different levels ofsample fluorescence. Note that gain settings do not increase the numberof photons detected but increase the output signal for a given photonlevel; increasing the gain increases the noise level.

Protocol 2. Assessing the optical performance of a confocalimaging system using subresolution fluorescentbeads

Equipment and reagents• 63 nm fluorescent microspheres (poly- • Confocal imaging system (e.g. the Bio-Rad

styrene Y-G 570) from Bangs Laboratories MRC600 CLSM): the system should have an« Glass coverslips (No. 1.5) electronic stepper motor to precisely con-. Silicone grease (Dow Corning, obtained tro1 focus position

from BDH Laboratory Supplies) • 1% polylysine solution

Method

1. Coat coverslips with polylysine by placing them in a 0.1% polylysinesolution for c. 10 min and then rinsing briefly with water. Removeexcess water before use.

2. Circle an area of the coverslip with a thin line of silicone grease andadd 50 ul of a 1:2000 aqueous dilution of the bead stock solution.Allow to settle for a few minutes before pouring off excess solutionand replacing with a further 100 ul water. Cover with a second cover-slip.

3. Place the sample on the microscope stage and move the required lensinto position. Set up the CLSM with the largest pin-hole size, high gainand high zoom (20), and begin scanning while focusing slowly up anddown until the beads are found.

4. Close down the confocal aperture to the desired level and focus on themedian focal plane of the bead (i.e. where the bead is widest). Set thelaser neutral density filter and photomultiplier gain to provide a bright,but not saturated signal, which does not cause excessive photobleach-ing of the dye.

5. Switch from normal (x-y) scanning mode to line scanning (x-z),Kalman collection filter (n = 3). Perform line scanning across thecentre of the bead using the stepper motor to 'section' in 0.1 um stepsfrom below the focal plane to above the focal plane of the bead. Theresulting image is the point spread function (p.s.f.) (57). Using aKalman collection filter makes the resulting image easier to interpret.Collect x-z images for five to ten beads.

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6. For each image plot fluorescence intensity against distance along aline through the centre of the image along both the x and z axes,respectively, and measure the distance between half-maximum inten-sity on each side of the maximum values. These are the full width half-maximum (FWHM) values and provide an estimate of the axial andlateral resolution, respectively. The values obtained from five to tenimages should be averaged.

9. Handling experimental material on the microscopestage

One of the principle requirements for intracellular ion imaging is the mainte-nance of cells in a healthy state yet be able to examine them under the micro-scope with appropriate access for dye loading or experimental manipulation.Several things need to be considered:

(a) Culture chambers. To provide the specialist culture requirements of somecell types, and to also allow experimental manipulation, a suitable culturechamber is essential. Chambers are available, or can be custom-built (41),to allow control of the medium composition (usually by perfusion of freshmedium through the chamber), temperature, and aeration. A problemwith culture chambers is that they can restrict access to cells, especiallyfor microinjection purposes. The contamination of chambers (particularlythose made of plastics) with chemicals such as ionophores, dyes, andinhibitors is a common problem, even after thorough washing. This is bestavoided by using disposable chambers or by keeping the same chamberfor the same chemical treatments.

(b) Movement of experimental material within the field of view. For cellswhich do not adhere inherently to the substratum, keeping a sampleanchored down to reduce the problems of vibration and movement duringimaging is a major issue. Methods for doing this include: adhering cells tocoverslips with polylysine (from Sigma) or Cell-Tak (from CollaborativeBioMedical Products); embedding cells in low temperature setting agar;or keeping cells in a minimal volume of medium. The use of a vibrationisolated workstation is also an important precaution to reduce specimenmovement. Holding cells in place can be a particular problem for micro-injection where direct access to the cells is required. Often cells may betrapped against a surface or held still with a suction pipette.

(c) Irradiation of cells. Cell irradiation for dye excitation is a serious cause ofphototoxic cell damage. Although irradiation (particularly from UVlight) may directly damage cells, the major problem is often photobleach-ing of the dye because this can result in the formation of toxic by-products

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and free radicals. This becomes apparent when the cytotoxic effects ofirradiation are compared for loaded and unloaded cells. The best ways tominimize this problem is to reduce the duration and intensity of irradia-tion, and the dye concentration.

(d) Temperature control. This can be achieved by several means, including:(i) monitoring and controlling room temperature with an air-conditioner,(ii) perfusing medium at a specific temperature through a culture chamber,(iii) using a culture chamber with a hollow 'jacket' through which water is

circulated from a temperature controlled water-bath,(iv) having a plastic hood mounted over the microscope or microscope

stage which allows the air temperature surrounding the sample to becontrolled (hoods of this type are available from several microscopemanufacturers).

(v) using a stage or culture chamber containing a resistive heater orPeltier device for temperature control. Changing sample temperaturein a controlled way for experimental purposes is even more difficultand this additionally requires measurements of stage or sample tem-perature (usually with thermocouples). It should be noted that evensmall changes in the temperature of the microscope stage can resultin changes in the focal position at the specimen.

10. Digital image processingThe image processing and analysis involved in quantitative ion imaging can beseparated into the four main categories listed below. Most commercial imageanalysis software packages are able to perform functions in the first two cate-gories (see Protocol 6).

(a) Numerical processing steps performed on the whole image, such assubtraction of dark or background signal or division of paired images(ratioing).

(b) Sampling regions of interest (ROIs) to obtain average pixel values.(c) Relating pixel values to ion concentrations by reference to calibration data.(d) Statistical analysis of image data, including: analysis of random noise in

pixel values within ROIs; comparisons of pixel values, or calibrated data,within or between individual images or populations of images.

11. RatioingRatioing in ion imaging refers to one or other of the following:

(a) The division of successive fluorescence images, recorded at the samewavelength, by the first image in the sequence (36).

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Richard M. Per ton and Nick D. Read

(b) The division of paired fluorescence images, recorded either s i imi lu inc-ously or sequent ia l ly at d i f f e r e n t e x c i t a t i o n or emission wave leng ths(32, 34, 35).

In the first case, ra t io ing can provide a use fu l visual guide for q u a l i t a t i v eimaging w i t h single wavelength dyes in order to i d e n l i f v changes in ion con-cent ra t ion , it usua l ly involves col lec t ing a sequence of images of a ce l l overl ime and then ralioint; each image aua ins t the first by a pixel-by-pixel d iv i s ionof each image pair (Protocol 3). Problems w i t h this approach arise if there arechanges in the dye d i s t r i b u t i o n , dye concentra t ion, fluorescence i n t e n s i l y (dueto photoblea.ehim;). or cell shape between successive images. The second easeis the more impor tan t and is what is most commonly refer red to as 'ratioimaging (Figure 6). R a t i o imaging of th is type invu lves the use of ratiometricdyes or ra l iomelr ic dye-pairs (Sect ion 4.3).

R a t i o imag ing wi th ni t iometr ic dyes or ra t iomelr ie dye-pairs can overeoinethe problems of v a r i a t i o n s in fluorescence brightness which are not due lo dif-ferences in ion c o n c e n t r a l i o n . These problems arise because of va r i a t i ons in:

(a) Dye c o n e e n l r a l i o n w i t h i n a cell or between d i f f e r e n t cells in a cellpopula t ion .

Figure 6. Manipulation of cytoplasmic pH in the apical regions of growing Neurosporahyphae (by weak acid and bast; treatments) and examinee) by dual emission confocalratio imaging of cSNARF-1. Celts were ester loaded with r.SNART-1 AM. Ratio imagescorrespond to median confocal optical sections through colls. (A) Hyphae ot Neurospurstreated with sodium benzoale 50 mM at pH 6. (B) Hyphae of Neurospora treated withtrimethylamine 50 mM al pH 7.8. Bar 10 um. Kindly provided by Sabine Fischer,

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(b) Cell thickness or thickness of the cell compartment loaded with dye.(c) Dye leakage from cells or dye redistribution between subcellular com-

partments.(d) Dye photobleaching.

These variables are cancelled out by division of images collected at twoappropriate wavelengths. Ratioing, therefore, allows considerable improve-ment in the quantification of ion concentration over that which can beachieved using single wavelength dyes (Sections 4.3, 12.4, and 12.5). However,ratioing does have its drawbacks, which are discussed in detail in Section 12.4.

Protocol 3. Producing a ratio image

Equipment• A suitable fluorescence imaging system • A microcomputer and associated software

(see Sections 6 and 7) for image analysis: the procedure described.For image collection a Bio-Rad MRC600 here was performed using TCSM (version 7)

CLSM was used software from Bio-Rad; all image process-ing was performed off-line

Method

1. Collect paired image data for ratioing (Sections 4.3 and 7.5) and saveas digital image files.

2. For each image of the image pair collect a 'background' image. Insome cases it is sufficient to interrupt the light path and collect the'dark' image produced by the detector in the absence of light. In othercases autofluorescence from the medium needs to be taken intoaccount so an image is collected from a nearby cell-free region. Themost difficult situation is where cellular autofluorescence has to becorrected for. Since imaging a cell before and after loading is notusually possible then an average or approximate autofluorescencevalue should be determined.

3. Correct each image of the image pair for background by performing apixel-by-pixel subtraction of either the corresponding dark or auto-fluorescence images. Alternatively subtract a standard value ('offset')from each pixel based upon the average autofluorescence.

4. Remove any remaining 'speckle' (resulting from random noise) fromoutside the imaged cell and any regions of low signal by either a'thresholding' or 'masking' step. Thresholding is performed by settingall pixels below a defined value to zero. The thresholding level isdictated by how noisy the fluorescence signal is; a thresholding valueof three times the noise level in the true fluorescence signal (3 x standarddeviations) is often used (see Section 12.2). Masking is performed bymultiplying the fluorescence images by binary mask images in which

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pixel values are either zero or one. Masks are usually produced by set-ting all pixels in the fluorescence image which are above a thresholdvalue to one and all below to zero. An alternative, less rigorous maskcan be produced from a bright-field image according to the cell out-line.

5. Define the relationship between ratio value and pixel value for theratio image. For 8-bit image data a ratio range of 0-2 corresponding topixel values of 0-255 is a useful starting point. However, wider ratioranges may be needed depending upon the range of ratio valuesexpected for the cell and the level of noise. The aim is to avoid exces-sive overflow where actual ratio values lie outside the defined range(some software programs give an indication of the extent to which thisoccurs).

6. Perform pixel-by-pixel division of the image pair. Programs for ratio-ing should interpret the division of a number by zero as zero ratherthan infinity. In some cases correction of minor misalignment in thecollected image pair may be made by shifting the position of one ofthe images relative to the other.

7. Perform any desired visual image enhancements such as pseudo-colouring, median, or smoothing filters, or conversion to 3D surfaceplots (Section 13.1).

12. Quantitative ion imaging12.1 Image quality and quantitative imagingImage quality for quantitative ion imaging may be considered in terms of theuseful numerical data which digital images contain. Important aspects relatingto quantitative ion imaging which need to be considered are:

(a) Precision of ion concentration measurement: the smallest differences orchanges in ion concentration which can be reliably detected. This ismainly limited by the random variation or 'noise' of the imaging system(Section 12.2). Precision may be increased by averaging the pixels withina ROI, or averaging or accumulating successive images over time. Deter-mination of the precision of ion concentration measurement for indi-vidual images allows meaningful statements about ion concentration tobe made at the individual cell level and facilitates comparison of datasampled from within the same image or pairs of images.

(b) Spatial resolution of ion concentration determination: the smallest areasover which ion concentration differences can be distinguished. This islimited by the areas over which image data is sampled in order to take

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into account random noise rather than the limits imposed by the optics ofthe system (41) (Sections 12.2 and 12.3).

(c) Temporal resolution of ion measurement: the speed with which imagescan be collected. Temporal resolution is largely dependent upon thedetector system used (Section 7) and upon the speed with which imagedata can be stored. If a video recorder is not employed then the speed ofdata storage is determined by the speed of the computer set-up used. Thequality and quantity of image data need to be balanced against temporalresolution. To improve temporal resolution, the quality and quantity ofimage data can be sacrificed by reducing image size, and thus the numberof pixels sampled, and decreasing the time over which photons arecounted. The highest temporal resolution can be achieved by fast linescanning in which a single line of pixels is recorded. This allows temporalresolution in the millisecond range (35).

(d) Reproducibility of ion concentration determination: the degree of agree-ment in ion concentration measurement for independent repeats. Repro-ducibility depends upon day-to-day and cell-to-cell variation in theimaging system and imaging data, and how well this is taken into account.The reproducibility of calibration procedures (which includes variationresulting from the preparation of ion concentration standards) is also afactor. Reproducibility may be improved at the level of image capture bystandardizing imaging settings and procedures.

(e) Accurate determination of ion concentration: the certainty in absoluteion concentration measurement. The degree of accuracy depends uponimage calibration and the reproducibility of calibration (Section 12.5).This dependence upon calibration makes ratio imaging much more reliablethan single wavelength imaging (Sections 4.3, 11, and 12.5). Independentverification of calibration data with an alternative technique of ionmeasurement (e.g. ion-sensitive microelectrodes) can be an important aidin demonstrating the accuracy of measurement (49, 69, 70). Note thataccuracy and precision are different. Results may be recorded with a highdegree of precision but, because of a systematic error in the measurementtechnique, still be inaccurate. In this respect all measurements of ionconcentration made by ion imaging methods should be considered asestimates of the absolute ion concentration.

There are many factors that operate both during image capture and sub-sequent processing, which can corrupt the integrity of image data (i.e. therelationship between pixel intensity and ion concentration or pixel position inthe image with respect to the region of the specimen actually sampled) result-ing in image 'artefacts'. Awareness of such potential problems is essential andcare is required in image capture, processing, and interpretation in order toavoid or take into account potential artefacts. Important sources of artefactsin quantitative ion imaging and suggested solutions are given in Table 4.

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Table 4. Summary of common ion imaging artefactsa

Problems Possible solutions

Optical distortions (in 2D and 3D) which 1. Use objectives appropriately correctedcan distort the width, shape, for chromatic and spherical aberrationand depth of the imaged field within a (Section 6.2).sample, and can misalign the two images 2. Use correct lens collar settings for coverslipused to obtain a ratio image (65-67). thickness.

3. Reduce refractive index mismatch alongoptical path by modifying refractive index ofculture medium or by using appropriateobjective immersion medium (ideally,for cells in aqueous medium use a waterimmersion objective).4. Use correction algorithms.

'Inner filter' and 'shading' effects 1. Avoid imaging deep within a sample.(excitation and emitted light attenuated or 2. Use multiphoton imaging.altered by passage through sample).Limited spatial resolution preventing 1. Use high numerical aperture objectivesaccurate determination of intracellular (Section 6.2).dye distribution. 2. Use confocal or multiphoton imaging

(Sections 7.3 and 7.4).Poor Rs/n as a result of random variation in 1. Maximize fluorescence signal detected by:pixel values within images (68). (a) increasing dye concentration;

(b) increasing intensity of excitation light;(c) using longer image integration periods or'accumulation' of successive images;(d) averaging successive images;(e) using wider band width filters;(f) opening confocal microscope pin-hole;(g) increasing detector gain.2. Use appropriate sampling strategies andstatistical analysis to take account of randomnoise (Sections 12.3 and 12.6).

a Note that imaging artefacts associated with properties of the fluorescent dyes are not included in thistable and are discussed in Sections 4, 5, 12.4, and 12.5.

12.2 Signal-to-noise ratioThere are different interpretations of the term 'signal-to-noise ratio' (Rs/n) inthe current literature. Here we define the Rs/n according to Sheppard (68), asthe ratio of the 'genuine' or 'useful' fluorescence signal and the variation(which may be determined as the standard deviation) (s.d.) in that signal. TheRs/n, provides a valuable measure of image quality in terms of the useful datawhich can be obtained. The Rs/n of an imaging system indicates its sensitivity(i.e. the smallest signal which can be reliably detected above the noise) andalso the level of precision of measurement possible (i.e. the smallest differ-ences which can be reliably distinguished).

Much of the confusion about the Rs/n arises from denning 'noise' in imaging.Noise is considered here to be the random variation within images. Noise

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originates from a variety of sources including: inherent, random, Poisson dis-tributed statistical noise (shot noise) associated with 'counting' photons;amplifier noise; digitization noise; detector dark current (signal in the absenceof light); cellular autofluorescence; stray background fluorescence; and evenout-of-focus fluorescence signal. It should be noted that the contributions ofthe detector dark current, cellular autofluorescence, and stray background flu-orescence to the overall recorded signal should be subtracted to leave the'genuine' dye signal (Protocol 3). These quantities themselves should not,therefore, be considered as noise. However, each is itself variable (due to shotnoise, amplifier noise, and digitization noise) and so subtracting the dark orbackground correction image from the overall recorded signal increases thevariation (noise) in the corrected signal. This variation is additive for the dif-ferent sources, degrading the Rs/n with respect to the true signal, thus:

where SR = overall recorded signal; Sg = 'genuine' signal; Sd = dark signal;Sb = background fluorescence; Sc = corrected signal = Sg; V = variation in,i.e. VSC = variation in corrected signal.

It is interesting to note that subtracting an average value, for example anaverage value of background signal, does not increase the variation in thecorrected image, whilst subtracting a variable background image does.

In an ideal system the only source of noise would be the Poisson distributedstatistical noise or 'shot' noise, which is inherently associated with 'counting'photons and sets the fundamental limit for the Rs/n (21):

Rs/n = useful photons counted/s.d. in photons counted = n/Vn = Vn.

As can be seen from this expression, with increasing numbers (n) ofphotons counted, Rs/n improves. More complex expressions for the overallRs/n, which include estimates of the effects of the other sources of noise, havebeen derived (68).

Experimentally, an estimate of the noise in an image may be obtained bycapturing the same image twice in rapid succession and determining the de-viation between corresponding pairs of pixels as described in Moore et al.(21). Alternatively, an even field of dye fluorescence may be imaged and, aftercorrection for background or dark signal, the standard deviation of pixel valuescalculated (note, for large numbers of pixels the distribution of the noiseapproximates to normal). As a general guide, the magnitude of the minimumdetectable signal (or difference in signal) needs to be at least three times thatof the noise level. The relationship between noise and precision of ion deter-mination is considered further in Sections 12.3 and 12.6.

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and


In confocal systems, because of the often reduced numbers of photonscounted, the Rs/n is often considered to be poor in relation to conventionalfluorescence imaging. However, if for non-confocal imaging only fluorescencephotons originating from the plane of focus are considered (i.e. the out-of-focus information (Section 7.2) is not included as contributing to usefulphotons counted) then confocal imaging compares more favourably withconventional imaging.

12.3 Numerical data extractionThe extraction of meaningful numerical data from images is a critical part ofquantitative ion imaging. Digitized images are two-dimensional arrays ofquantitative values organized in discrete pixels (or voxels—the 3D equivalentof a pixel). It is recommended that the actual pixel size (with respect to thespecimen) should be set at a third of the optical resolution of the imagingsystem, according to the Nyquist sampling theory (71). The values of pixelstake the form of 'grey levels' and are generally between 8-bit (256 grey levelsor 28) and 32-bit (232 grey levels). The number of grey levels determines howmuch and how accurately information can be recorded in the image.

In principle, the value of each pixel relates to the number of photonsdetected from that region of the specimen and, in the case of ion imaging, tothe ion concentration. However, because of the random variation betweenpixels (Section 12.2), each pixel in an image does not accurately represent ionconcentration and examining numerical data at the individual pixel level canbe misleading. To take account of this noise, ROIs are 'extracted' and anaverage value obtained (41). Two important considerations for ROIs are:

(a) The size of the ROI (number of pixels) sampled. The size of area fromwhich an average value is extracted imposes a limit upon the spatialresolution of numerical data. It is this rather than optical resolution limitswhich determine spatial resolution in ion imaging. The number of pixelssampled determines to what extent random pixel variation can be takeninto account and so affects the precision with which changes or differencesin ion concentration can be determined. In this way the spatial resolutionlimit, the degree of noise in the image, and precision of ion measurementare all interdependent. Dye sequestration within subcellular compart-ments is another factor to consider when deciding upon sample area size.Where dye sequestration occurs some averaging may be unavoidable evenwith small sample areas.

(b) Deciding where to place ROIs within the image. In addition to the prob-lem of dye sequestration within different subcellular compartmentsdescribed above it is also important to avoid regions of ambiguous signal,such as occur at the extreme periphery of cell boundaries or regions oflow signal and high noise. Many software packages offer the possibility ofdefining hand-drawn or regular-shaped sample areas over one's images.

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6: Calcium and pll imaging in living cells

Although the former may he convenient for s e l e c t i n g precise regions, thel a t t e r arc hel ler lor s landard ized . repeated sampling at a known spatialresolution (n X n p i x e l s ) .

It is i m p o r t a n t to s ia tc the optical resolution, the pixel dimensions (deter-m i n e d by the op t ica l magnification. electronic zoom, and the detector). andthe numbers of pixels sampled over when repor t ing data extracted fromimages.

12.4 Quantitative ratio imagingA d i s t i n c t i o n should be made between qua l i t a t ive and q u a n t i t a t i v e rat ioimaging . A l t h o u g h ratio images, formed by p ixe l -by-p ixe l division of thee n t i r e fluorescence image pair, arc often displayed, t h e i r importance is v isual(Figure 6, Figures 7A and 7B. Protocol 3) and they should not he used forsubsequent numerical analys is . Ins tead iiumerical data should be extractedfrom corresponding ROls in the ind iv idua l lluoresccnce images of ratio pairsand the average values of the sampled ROls divided (Figures 7A and 7B'Protocol 4). The reason for th is is tha t there is a noise-dependent difference inthe average r a t i o value when this is determined d i r e c t l y from a rat io imager e l a t i v e to the division of two average fluorescence values from the f luores-ccnce image pair. As the noise in the fluorescence images increases the d i f fe r -ence between these average r a t i o values also increases. This difference

Figure 7. Quantitative analysis of cytoplasmie pH in the apical regions of a growingNeurospora hypha and Dryopteris rhizoid performed by simultaneous dual emission con-focal ratio imaging of cSNARF-1. (A) Ratio image of a hypha pressure injected with10 kDa cSNARF-1 dextran. (B) Ratio image of a Dryopteris rhizoid ester loaded withcSNARF-1 AM. (A') and (B r) Graphs of pH v. along a inidline through the cells shown in thecorresponding (A) and (B). A pseudocolour scale is shown with corresponding pH valuesfrom an in vitro calibration (MES/Hepes buffer). Bars = 10 um. Modified from ref, 30 withpermission.

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becomes more significant when attempting to obtain very precise measure-ments of ion concentration with high spatial resolution (30).

Protocol 4. Quantitative ratio analysis

Equipment• A computer, associated software, and frame poration); and Excel 7 (Microsoft) on images

store for image capture, processing, and captured on a Bio-Rad MRC600 CLSM (Bio-analysis. The power of the computer, the Rad PIC format files). All image analysisimaging software, and frame store used described is performed off-line. All proce-determine the rate at which ratioing can be dures may be performed using appropriateperformed. The procedures described are alternative programs,performed using Optimas 5 (Optimas Cor-

Method

1. Follow Protocol 3, steps 1-4. Equivalent processing may be donedirectly in Optimas 5 after converting the Bio-Rad PIC format imagesto TIFF format.

2. Extract average fluorescence values from defined ROIs at correspond-ing positions in the paired fluorescence images using Optimas 5 andcalculate the ratio of these average values. (How to choose the size ofROIs and where they should be placed within the fluorescence imagesare discussed in Section 12.3.)

3. Extract individual pixel values from the defined ROIs within the pairedfluorescence images (a macro to perform this can be provided byOptimas user support) and import into a spread sheet (e.g. Excel 7).

4. Determine the degree of variation (i.e. the standard deviation) in thepixel values within ROIs using the facilities available in Excel. Fromthis the signal-to-noise ratio can be determined (Section 12.2). Morecomplex statistical analysis can also be performed, as discussed inSection 12.6, to quantify the effect that variation in the fluorescenceimages has on the precision of determining the final ratio value.

5. Relate the ratio values to ion concentration according to the experi-mental procedure described in Section 12.5 and Protocol 5. Analysisof the variation in ratio value for ROIs of different sizes (n x n pixels)provides estimates of the precision and spatial resolution limits towhich ion concentration can be measured (see Figure 8 and Section12.6).

Although ratio imaging potentially provides the most accurate and precisemeans of quantitative ion imaging, it is not without its problems which canlead to serious errors in absolute ion concentration measurement if they arenot taken into account. In addition to the detrimental effect of noise in theindividual fluorescence images used to generate the ratio data (see above),

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the main problems affecting the relationship between ratio value and ionconcentration are:

(a) Temporal misalignment of the fluorescence image pairs.'This is generallya problem associated with the sequential capture of fluorescence imagesfor ratioing. Problems arise when cell movement or growth occurs betweencapture of the first and second image of a ratio pair resulting in mismatchof the two fluorescence images. This places significant demands upon therate of image capture, necessitating:(i) Rapid changes in excitation filters (for dual excitation dyes) or in

emission filters (for dual emission dyes).(ii) Rapid sequential image capture and saving to computer disk. Simul-

taneous dual wavelength excitation imaging and/or dual wave-length image capture is possible in some cases (Sections 4.3 and 7.5;Figures 6 and 7) and can provide a solution to this problem.

(b) Positional misalignment of the image pair. This problem can occur withtwo images acquired simultaneously by dual channel imaging if the twodetectors are misaligned, causing misregistration of the two fluorescenceimages in the x-y plane. Additionally, chromatic aberration can causemisalignment in the z plane of both sequentially and simultaneouslycaptured images where the two wavelengths differ significantly. To avoidthese problems, precise optical alignment of the detector system, andcorrection for the different wavelengths used, are necessary.

(c) 'Bleed-through' ('cross-talk') of fluorescence between detector channels.Bleed-through occurs when there is incomplete discrimination betweenthe emission spectra of the two dye forms (ion bound and ion free) of aratiometric dye or the two dyes of a dye-pair. This problem is most signif-icant in the latter case when using two dyes for ratioing (Section 4.3). Theextent of contamination between imaging channels should be checked bymeasuring the fluorescence signal in both imaging channels from each dyeimaged on its own. Changing the filter set, changing the relative proportionof each dye in the mixture, or the detector gain settings of each imagingchannel may correct the problem. Alternatively, a correction equationmay be derived empirically by examining the degree of bleed-through atvarious dye concentrations (64).

(d) Autofluorescence differentially contaminating different channels. Theproblem may be resolved by a similar approach to that discussed in thelast point by using appropriate filters or gain settings.

(e) Differential rates of photobleaching of the two forms of a ratiometric dyeor the two dyes of a ratiometric dye-pair. Although this would be ex-pected to be more of a problem with ratiometric dye-pairs it has beenshown to be a problem with the ratiometric dye Indo-1 (19). Dye photo-degradation should be minimized by reducing excitation illumination.

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(f) Differential rates of dye leakage from the cell or sequestration withinorganelles of the two dyes of a ratiometric dye-pair. This has been over-come in the case of Calcium Green-Texas Red (Table 1; Figure 1E)where the two dyes are co-conjugated to a 70 kDa dextran.

12.5 Calibration of dye responseOne should be aware that, although the ultimate aim of quantitative ion imag-ing is to obtain absolute values of ion concentration, the actual valuesobtained are, in reality, only estimates. How well these estimates reflect thetrue ion concentration values is limited primarily by how the dye behaves inthe intracellular environment, and how well the dye response is calibrated.

The relationship between fluorescence emission and ion concentration maybe described in terms of the equilibrium equation for the binding of dyemolecules to free ions. The following equations apply for 1:1, dye:ion interac-tion, as occurs with Fluo-3 or Fura-2 (31, 72).For a single wavelength dye:

[ion] = Kd (dye-ion complex)/(free dye) = Kd (F - Fmin)/(Fmax - F)

Similarly, for a ratiometric dye:

[ion] = Kd F2free/F2bound (R - Rmin)/(Rmax - R)

where Kd = dissociation constant; F = measured fluorescence; Fmin = the fluor-escence when all dye is uncomplexed, i.e. at zero [ion]; Fmax = the fluor-escence when all dye is complexed with the ion, i.e. at saturating [ion]; R =the ratio of fluorescence intensity at wavelength 1 divided by the fluorescenceintensity at wavelength 2; Rmin = fluorescence ratio at zero [ion]; Rmax =fluorescence ratio at saturating [ion]; F2free = dye fluorescence at wavelength 2with zero [ion]; F2bound = dye fluorescence at wavelength 2 with saturating[ion]. For derivations see Grynkiewicz et al. (31) and Thomas and Delaville(72).

Calibration is primarily concerned with the determination of the .Kd valueof the dye and the values of Fmin and Fmax (or Rmin and Rmax for a ratio dye).These values must be determined experimentally; published Kd values shouldbe used with caution (20, 73). The Kd values of ion-sensitive dyes are usuallyhigher in vivo than in vitro (17). With single wavelength dyes, Kd values areusually determined in vitro by imaging a series of dye buffer solutions over arange of known ion concentrations. Often a modified medium is used with ionconcentrations (e.g. Mg2+, K+, Na+, etc.), viscosity, and hydrophobicityadjusted in an attempt to mimic the intracellular environment (24, 72). The Kd

is rarely determined in situ as this requires that there is no change in intra-cellular dye concentration during the calibration procedure, a situation whichis difficult to guarantee. As a result of the differences in the dye content ofindividual loaded cells (Section 5.8), meaningful calibration for a particularcell necessitates the determination of Fmin and Fmax for that individual cell, in

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situ. These difficulties often restrict the use of single wavelength dyes to thequalitative analysis of changes in ion concentration. Kd values for ratiometricdyes can also be determined by in vitro experimentation. However, with suchdyes the problem of variations in intracellular dye concentration is less signifi-cant. Kd values are, therefore, commonly determined from a three point insitu calibration (Protocol 5). This involves recording ratio values at zero andsaturating ion concentrations (Rmin and Rmax, respectively) and at an inter-mediate ion concentration and then fitting the data to the equation above.Kd(F2free/F2bound) is sometimes determined experimentally as a combined con-stant. Note that when wavelength 2 is the ion-insensitive wavelength (isos-bestic point) then F2free equals F2b0und so Fatree/F2bound = 1. The calibration ofratiometric dye-pairs which consist of two ion-sensitive fluorochromes (Sec-tion 4.3) is complicated because two different Kd values for Ca2+ binding areinvolved. This means that the standard equations for calibration are no longervalid, although experimentally derived calibration curves can be used.

12.5.1 Calibration strategies for calcium and pH imagingThe ability to obtain good calibration data is severely hampered because dyebehaviour varies under different environmental conditions (see Section 4.2).Due to the lack of knowledge about the dynamic physical and chemical natureof the intracellular environment, it is very difficult (if not impossible) toreproduce such conditions in vitro. As a result, experimenters often strive tocalibrate dyes in situ by, for example, using ionophores in conjunction withexternally applied solutions of known ion concentrations to clamp the intra-cellular ion concentration to defined values (Table 5 and Protocol 5). Detailedprotocols for in situ calibration procedures may be found in Thomas andDelaville (72). Problems with these methods include:

(a) Unstable clamping of cytoplasmic ion concentration, often because of theactivities of cellular homeostatic mechanisms. Procedures sometimesincorporate metabolic or ion pump inhibitors to combat this.

(b) Rapid loss of dye or sequestration within organelles which can causeapparent shifts in cytoplasmic ion concentration.

(c) Cellular disruption and alteration of the intracellular environment.Although in situ calibrations are sometimes referred to as in vivo calibra-tions, this is not always an appropriate term as the effects of ionophoretreatment and disruption of the intracellular ion concentration tend to betoxic with effective clamping.

Because of the difficulty in interpreting the results obtained by in situmethods is in genuinely fixing the cell at the desired ion concentration the bestcalibration data incorporates an independent method of ion concentrationmeasurement such as ion selective microelectrodes (49, 69, 70).

Multiple point calibrations, which allow accurate curve fitting to the data,

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Table 5. Summary of techniques used for calibrating ion imaging data

Types of calibrations Result Advantages/problems Ease

In vitro

Buffer solutions:- MES/Hepes for pH-Ca2+/EGTA for Ca2+

'Pseudocytosol' buffersolution (24)

In situ

lonophorepermeabilization of cells(47, 68; Protocol 5)

Cell permeant weak acidsand bases (e.g. propionicacid and trimethylamine) (13)Combined use of dyeimaging and ion-sensitivemicroelectrodes(Sections 12.5.1 and 14)

Multiple point A good standard for comparison but Very easy,response curvea the Kd of the dye in vitro often varies routine

from that in vivo (Sections 4.1 and 4.2). Relatively easy.Multiple point A basic estimation of absolute [ion]1.b routineresponse curve

Single pointto Intheory, the best estimation of absolute Difficult toa few stepped [ion],. Problems with incomplete clamp achieve goodchanges of [ion]i, cellular disruption/toxicity, loss results; set-up

of dye, and dye compartmentalization. is not routineAs above Problems as above, especially incomplete Easy to perform

clamp of [ion],. Only useful for pH. but generallyunsatisfactory

Single point to Requires complex apparatus and Technicallya few stepped technical expertise but potentially demanding; notchanges the best method—[ion], need not be routine

clamped to specific values.

a Response curve = dye fluorescence (or ratio) versus ion concentration.b(ion]( = intracellular ion concentration.


are desired. Where a full dye response curve is obtained, calibrated valuesmay be read directly without calculating the Kd, Rmax, and Rmin- Often, how-ever, two or even one point calibrations are all that are possible during anexperiment. In such cases the dye response over the rest of the ion concentra-tion range may be inferred by reference to in vitro data.

Protocol 5. In situ calibration

Note that this protocol is a generalized description of the use of iono-phore or protonophore methods for in situ calibration. The procedure isof most value with ratio imaging techniques where the result may beapplied to the entire population of cells if standard imaging settings areused throughout. With single wavelength imaging calibration must beperformed for each cell imaged.

Equipment and reagents• Incubation chamber allowing access to

cells, preferably with provision for perfu-sion of medium (Section 9)

• Equipment for dye loading, if necessary(see Sections 5.5-5.7)

• Fluorescent dye• lonophore or protonophore: ionomycin

(Sigma) or Br A23187 (Molecular ProbesInc.) for Ca2+ and nigericin (Sigma) ornigericin/valinomycin/K+ for pH (17, 72, 74).Different cell types exhibit varied responsesto different ionophores so it is advisable totry different ionophores and various con-centrations (generally over the range 10 uMup to mM concentrations).

• MES (Sigma)

> A suitable fluorescence imaging system(see Sections 6 and 7)

• Hepes (Sigma)• EGTA (Sigma)• Calibration media (which may be based on

the standard cell culture medium) with theion of interest buffered at known concentra-tions. Where the ionophore used is knownto allow the transport of other ions, theconcentration of those species should befixed at the concentration expected withinthe cell. It is also useful to regulate the ionicand osmotic strength of the calibrationmedia over the range of different ion con-centrations used.

Method

1. Prepare calibration media. pH can be buffered over the range 5.5-8.5using 10 mM MES/Hepes whilst Ca2+ concentration can be controlledwith 10 mM EGTA mixed with varying concentrations of CaCI2 (75).Temperature can affect both the buffers used to control ion concen-tration and the dyes used for imaging and should also be taken intoaccount.

2. Prepare cells in the culture chamber for loading and treatment. Cellsshould be adhered to the substratum or held in position (Section 9) toprevent movement during the changes in calibration medium.

3. Load cells with dye (Section 5).

4. Collect the required experimental imaging data.

5. Apply calibration medium buffered at the first ion concentration inthe calibration series in the presence of ionophore and allow cells toequilibrate. Images should be collected at intervals until a steady

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state is reached. Experience will reveal how long equilibration shouldtake and whether a reliable steady state can be reached before declin-ing cell health and image quality cause the observed values to change.

6. Apply the second calibration buffer and repeat the imaging timecourse. Continue the procedure with further ion concentration incre-ments covering at least minimal (effectively zero) free ion concentra-tion, dye saturating ion concentration, and an intermediate value.

7. If cells lose fluorescence or become significantly disrupted during theclamping procedure it may only be possible to impose one ion con-centration change. This is a severe limitation when using single wave-length dyes. However, in the case of ratio imaging, the calibration maybe continued by repeating the experiment with fresh cells for each ionconcentration buffer in the calibration series.

8. Finally, if possible, return cells to the initial ion concentration calibra-tion buffer. The value obtained should be the same as that recordedoriginally.

9. Perform a simple in vitro calibration with free dye in the media usedfor in situ calibration. Use the appropriate form of the dye in the invitro calibration (i.e. free acid or dextran conjugate). This provides areference to compare calibrations on different days with differentinstrumental settings.

10. Construct calibration curves (see Figure 8} by plotting data points orfitting the data to the theoretical curve described by the equationsgiven in Section 12.5. The ion concentration may be derived directlyfor the calibration curve or calculated using the appropriate equation.

12.6 Statistical analysis of image dataStatistical analysis of quantitative image data is often confined to the analysisof variation in measured ion concentration (after suitable calibration), withinpopulations of cells or between cell populations given different experimentaltreatments. This generally involves using simple methods such as the studentst-test (for confidence intervals and comparison of means). Such an approachtends to ignore the variation in the pixel data within the individual digitalimages. However, as the limits of quantitative imaging are pushed to theirextreme (in order to make more precise measurements of ion concentrationwith better spatial resolution) the need for adequate analysis at the pixel levelbecomes more pressing.

Statistical analysis of pixel value data has important applications in:

(a) Examining the inherent variation in image data (particularly in relation tothe Rs/n) in order to define the limits of precision and resolution for ion

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Figure 8. Example of an in vitro calibration curve for cSNARF-1 free acid with error barsindicating the precision of measurement. Spatial resolution was determined by the 14x14pixel sampling area size (ROD. Error bars in all graphs are the 95% Bayesian interval forthe mean ratio of the small sample areas based on variation between individual ratiovalues of each pixel (see Sections 12.3 and 12.6 for further explanation). Modified fromref. 30 with permission.

measurement (see Sections 12.2 and 12.3; Figure 8) (30). Image collectionparameters and image sampling can subsequently be optimized withrespect to the precision and resolution of ion measurement necessary toallow meaningful biological interpretation of the data.

(b) Analysis of spatial inhomogeneities in ion concentration within individualimages (of individual cells). A good example of this is in investigating thepresence of intracellular ion concentration gradients associated withpolarized cell growth (30, 76).

(c) Analysis of ion concentration changes between images of individual cellscollected sequentially in a time course.

(d) Comparison of differences in ion concentration in individual cells of a cellpopulation. This allows the heterogeneity in individual cellular responsesto be recorded. If data is averaged across cell populations rather thanbeing considered at the single cell level such heterogeneity is lost.

(e) Distinguishing between pixel variation within images (a fundamentallimitation imposed by the imaging system), cell-to-cell variation within a

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uniformly treated cell population, and differences between experiment-ally treated and untreated cells. Statistical methods (e.g. analysis of vari-ance testing or non-parametric tests) (77) are available to analyse thecontribution of each source of variation to the overall observed variation.

Image data can pose particular problems for statistical analysis:

(a) Extraction of individual pixel values from digital files, which is not avail-able as a standard function in many image analysis software packages(Protocol 4).

(b) Problems related to sampling pixel data from defined ROIs within digitalimages of cells for analysis of pixel variation. Images of cells, unlike'ideal' in vitro test images, are subject to additional sources of pixel varia-tion including regions of different dye concentration and different ionconcentrations. How such variation is recorded is highly dependent uponboth the size and placing of ROIs within an image (Section 12.3).

(c) The statistical nature of the inherent variation in photon counting ('shotnoise')—which is Poisson distributed (Section 12.2). This dictates that thevariation in the fluorescence signal is proportional to the mean fluores-cence intensity and so, due to the relationship between fluorescenceintensity and ion concentration, the precision of measurement itself varieswith ion concentration.

(d) The propagation of variation initially present in a captured image duringsubsequent image processing steps, such as image subtraction and divi-sion (Section 12.2).

Standardizing image collection with respect to average fluorescence intensity(and Rs/n) by using standard imaging conditions and image collection para-meters, simplifies statistical analysis and allows more rigorous comparison ofdata. The inherent variation between pixels due to noise generated by theimaging procedure may be investigated with different image collection para-meters by examining test solutions (uniform dye/buffer mixtures) of knownion concentration in vitro. These results can be compared with the betweenpixel variation observed in vivo, within individual images of dye loaded cells.This variation is likely to increase with uneven dye distribution and specimenthickness, dye sequestration within organelles, and local domains of differention concentration.

Ratio imaging poses a significant problem with regard to statistical analysis..In ratio imaging, the average ratio must be calculated by dividing averagefluorescence intensity values of images collected at the two wavelengths (seeSection 12.4 and Protocol 4). Thus, statistical analysis cannot be performeddirectly on the ratio image pixel data. The pixel variation within the fluor-escence images should be considered and for this to be done correctly therelationship between individual corresponding pairs of pixel values (covari-

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ance) needs to be determined. This requires more advanced statistical analy-sis, for example the Bayesian approach employed by Parton et al. (30). Withmore complex problems such as these, expert statistical advice should besought.

13. Visual presentation of image data13.1 Visual image enhancementThere are various ways in which digitized images may be visually enhanced.The most important for ion imaging are pseudocolouring and 'smoothing'(using the various smoothing, averaging, and median filters available withmany image processing software packages).

Pseudocolouring, assigning pixels different colours according to theirnumerical value, is particularly useful in drawing attention to small or local-ized differences and is routinely applied to images of ion distribution, usuallyratio images. A pseudocolour scale with a specific 'look-up table' (LUT) canbe easily custom-designed with most imaging software. However, pseudo-colouring should not be over-interpreted. Reference to the pseudocolourscale may give the impression that individual colours or hues always representspecific values of ion concentration. This is usually incorrect due to therandom variation in pixel values inherent in images (Section 12.2) and the factthat LUTs are often stepped such that a range of pixel values correspond to agiven colour. An alternative to pseudocolouring which is commonly used todraw attention to spatial differences in images is three-dimensional graphicalrepresentation, in which pixel intensity is coded as height to give a three-dimensional surface (36). This is, however, only available with some softwarepackages (such as NIH Image).

Smoothing, averaging, and median filtering makes noisy images look moreeven and is useful for reducing distracting random 'speckle' for qualitativeexamination. However, caution should be used because of the way in whichfilters can affect boundaries, such as the extreme periphery of a cell, or distorthighly localized changes in pixel value. Filtering should not be applied beforeextraction of numerical data for quantitative analysis unless one is fully awareof how the pixel values are mathematically transformed and the likely effecton quantitative results.

13.2 Preparation of digitized figures and plates forpublication

As ion imaging deals almost exclusively with digital image files it is convenientto avoid traditional photographic processing of figures for publication andwork digitally. Protocol 6 describes the preparation of figures and plates in dig-ital form for submission to journals either as hard copy or in digital formatsaved on disk. When submitting images or composite plates on disk it is

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essential to also submit top quality hard copies. This is because variationbetween the calibration of the monitor/printer of the publisher and the systemon which plates were initially prepared can cause deviations from the expectedcolour shades and the brightness and contrast of grey scale images. It is alsouseful to supply individual TIFF files of the images. If problems arise with thecomposite file format or with reproduction of either images or text, the pub-lisher can then remake the plate from the TIFF files and add labelling by refer-ence to the hard copies. Where possible a modular design in composite platesshould be used (see Figures 6 and 7) because this can be more easily dissectedby the Publisher if rearrangement is necessary for space in the journal.

Printers for generating hard copies of figures/plates (e.g. dye sublimationprinter, video printer, ink jet printer, or laser printer) vary considerably in theprices of hardware and media and the quality of printouts. We recommendthe latest generation of ink jet printers as both the hardware and media areextremely cheap relative to other systems and both grey scale and colourreproduction is good enough for most applications except where the highestquality is required (Protocol 6).

Protocol 6. Preparation of digitized figures and plates forpublication

Note that although Macintosh systems are most commonly used by Pub-lishers, PC compatible computers are still more common amongst re-searchers. This protocol is based on the use of PC compatible softwarepackages and equivalent Macintosh software packages for particular pro-cedures are given.

Equipment• Digital image files for processing (e.g. Bio-

Rad PIC files collected using the Bio-RadMRC600 CLSM and COMOS 7.0 software)

• Personal computer with sufficient RAM (> 8Mb) and processor power (pentium 120MHz or better) to handle image data. Alarge hard drive (500 Mb-1 Gb) or archivingsystem (e.g. a magneto optical disc drive orwritable CD ROM) is advisable for storingimages.

• Hewlett Packard Desk Jet 850 colour ink jetprinter and Hewlett Packard (HP) PremiumGlossy paper

• Software for image processing and addi-tional functions. The packages used rou-tinely by us are: Microsoft PowerPoint 7.0;PaintShop Pro 4.0 (JASC Inc.); Core/draw3.0 (Corel Corp.); Confocal Assistant 4.02;Optimas 5.0 (Optimas Corp.); Fig. P 2.7(BioSoft). (Roughly equivalent Macintoshprograms are: Adobe Photoshop 3.0;Adobe Acrobat Pro 2.0; Adobe Pagemaker6.0; Adobe Illustrator 6.0; NIH (NationalInstitutes of Health) Image and Sigma Plot(Jandel), respectively.)

Method

1. Open the Bio-Rad PIC file in Confocal Assistant 4.02, then open the de-sired colour look-up table (LUT), resave and convert to tagged imagefile format (TIFF) which is the preferred format for image handling (usethe RGB variety of colour TIFF).

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2. Import the TIFF file into PaintShop Pro 5.0. The TIFF file may then becropped to size, rotated, or resized as desired. Scale bars may beconveniently added into the TIFF file at this stage. Digital images aregenerally collected at a limited resolution of (between 512 x 768 to1024 x 1024). To improve this, for high quality reproduction, TIFF filesshould be saved at high resolution in Paint Shop Pro 5.0 (save as:options; set 'dots per inch' DPI to 1400). For submitting digital imageson disk to a journal the resolution required is usually specified by thePublisher.

3. Import the processed TIFF file(s) into Powerpoint 7.0.

4. To produce a composite plate which contains line drawings andgraphs, these should be prepared using suitable software (e.g. Corel-draw 3.0 for line drawings and Fig. P 2.7for graphs) and imported intoPowerpoint 7.0.

5. Arrange images, diagrams, and graphs and add labelling.

6. Produce high quality hard copies using the Hewlett Packard Desk Jet850 colour ink jet printer on HP Premium Glossy paper. The best greyscale and colour reproduction is obtained when the printer has beencalibrated specifically for the attached computer and monitor.

14. Combining ion imaging with other experimentaltechniques

Besides combining imaging with simple treatments involving the externalapplication of different environmental stimuli, agonists, inhibitors, and ionchelators it is often highly desirable to also combine these approaches withmore sophisticated experimental techniques or other experimental methodsfor the measurement of ion concentration or ion channel activity. Importantexamples include:

(a) The use of caged compounds (78). A caged compound is usually a bio-logically important ion or molecule (e.g. Ca2+, H+, cAMP, inositoltriphosphate, Ca2+ buffers, or neurotransmitters) bound to a photolabile'cage' which masks its biological activity. Irradiation of the cage with UVlight (photolysis) releases the bound species in its active form. Cagephotolysis is usually achieved with UV light irradiation from the fluor-escence excitation source or with specialist UV flash photolysis systems(18). Alternatively, irradiation with the UV or IR laser of a UV CLSM ormultiphoton laser scanning microscope (Sections 7.3 and 7.4) can allowhighly localized photorelease of caged compounds. Caged moleculesmay be loaded into cells by similar means as used for fluorescent dyes(Section 5): microinjection or electroporation of cell impermeant forms;

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ester loading of the AM ester forms (e.g. nitr-5-AM or diazo-2-AM); and,in the case of the cell permeant caged cyclic nucleotides, by direct diffusion.Caged compounds provide a relatively non-invasive means to manipulateintracellular signalling processes in a very specific and controlled mannerwhich is often highly localized in time and space within living cells.Although intracellular Ca2+ or H+ are often imaged in combination withcaged probe release, UV excited ion-sensitive dyes (e.g. Fura-2 and Indo-1) are generally not used for this purpose because of the possibility of dyephotobleaching during cage photolysis.

(b) Microinjection of molecules (such as calcium ion buffers) (25). Pressureinjecting the calcium buffer BAPTA has been used to collapse intra-cellular calcium gradients to investigate their role in tip growth (25). Arange of other molecules can also be introduced into cells by pressuremicroinjection in order to experimentally manipulate signalling pathways,including proteins such as calmodulin.

(c) The manganese quench technique (79). Manganese ions (Mn2+) can act asa surrogate for Ca2+ and are taken up into by cells through calcium chan-nels. Unlike Ca2+, however, Mn2+ causes fluorescence quenching of theCa2+-sensitive dye Indo-1 upon binding to it. Non-inhibitory concentra-tions of Mn2+ (~ 200 uM) are applied externally to Indo-1 loaded cells andchanges in fluorescence are imaged at the Ca2+-insensitive emission wave-length (450 nm) over time. This allows the occurrence of localized Ca2+

channel activity in the plasma membrane to be mapped and measured.

(d) Simultaneous electrophysiological measurements (69). It is possible tosimultaneously measure membrane potential and/or ion channel activityand intracellular ion concentrations whilst imaging intracellular pH orCa2+ ion concentration. This combination of electrophysiological andimaging techniques can provide invaluable information on the interrela-tionship between signalling processes. The main problem with such anapproach is in the elimination of electrical interference, which originatesfrom the imaging system and external sources, and which adversely affectselectrophysiological measurements. A wire mesh Faraday cage needs tobe fitted round the microscope for this purpose (35). Autofluorescencefrom the microelectrodes or patch pipettes used for electrophysiologicalrecordings can also cause problems and needs to be taken into account.

15. Critical controls for intracellular ion imagingIt is appropriate to conclude by summarizing the most critical controls whichshould be carried out routinely as part of any ion imaging procedure:(a) Intracellular dye distribution. Bright-field, phase-contrast, or DIG micro-

scopy should be used to image dye loaded cells allowing dye distribution

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to be mapped and correlated with cytological features. Ideally, confocalfluorescence microscopy should be used to assess the extent to which dyeis sequestered within organelles during the time over which ion imaging isperformed.

(b) Cell health. Although cells may remain viable throughout an imaging pro-cedure, their normal activities may be significantly perturbed. It is, there-fore, important to determine whether cells behave normally over theduration of an imaging experiment. Following cellular morphology,growth rates, or response times in dye loaded cells during imaging andcomparing these with the same parameters examined in unloaded cellsprovide the easiest measure of cell health. Possible perturbatory effects ofculturing cells on the microscope stage (Section 9), the dye loading pro-cedure (Section 5), the presence of intracellular dye, and the cytotoxiceffects of dye excitation (especially with laser or UV light) all need to beassessed.

(c) Intracellular dye response. Imposing changes on the intracellular ion con-centration, as with in situ calibration (Section 12.5 and Protocol 5) is themost common method of checking the responsiveness of intracellular dye.In some cases the dye may become chemically altered (e.g. by photo-damage) or irreversibly bound to cell walls or membranes such that ionconcentration in the cell compartment of interest is no longer reported.

(d) Routine performance checks on the imaging equipment (see Section 8and Protocol 2). In addition, performing a simple in vitro calibration aftereach experiment allows systematic errors (such as incorrect setting up ofthe equipment on a particular day) to be detected.

AcknowledgementsThanks to Dr Mark D. Fricker for informative discussions and advice and alsoto Sabine Fischer for her help in preparing the manuscript.

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274

Reflection-contrast microscopyJ. S. PLOEM, F. A. PRINS, and I. CORNELESE-TEN VELDE

1. Introduction1.1 MethodologyFaure-Fremiet (1) used reflected light in cell biology studies half a centuryago. Curtis (2) and Izzard and Lochner (3) performed the first fundamentalstudies of interference images of living cells on glass surfaces using reflected-light microscopy. Reflection images were obtained with scanning reflectingmicroscopy (4, 5). Reflected-light microscopy for the study of living cells (2, 3,6, 7) has been described as interference reflection, reflection-interference con-trast, surface-contrast, and surface-reflection interference microscopy (8).Ploem (9, 10) investigated further optical methods to improve the image con-trast in reflected-light microscopy. In collaboration with Leica (11-13), animproved microscope system was developed for reflected-light microscopy. Itused an aperture diaphragm with a central stop (creating an annular aperture)in the epi-illumination light path at an aperture plane conjugate with the backfocal (aperture) plane of the objective (14). This was combined with epipolar-ization microscopy using immersion objectives equipped with a quarterlambda plate in their front lens (15). New high aperture objectives were de-veloped for high resolution biological microscopy. This optically improvedversion of reflected-light microscopy was named reflection-contrast micro-scopy (RCM).

1.2 ApplicationsWith RCM, a considerable improvement of image quality has been obtainedwhich allowed the observation of weakly reflecting microscopic structures.Most of the early applications of RCM were directed towards the observationof living cells (16). A significant improvement of the image contrast in RCMwas obtained in 1982. Bonnet observed (unpublished) that oxidized polymer-ized diaminobenzidine (DABox), the end-product of an immunoperoxidasestaining, showed very strong reflectance. This finding was used for a non-radioactive in situ hybridization with peroxidase-DAB as label and RCM asthe microscopic detection technique (17, 18). It resulted in the first successful

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J. S. Ploem, F. A. Prins, and I. Cornelese-ten Velde

detection of a unique gene on a human metaphase chromosome using a non-radioactive method. Hoefsmit (19) described early applications of RCM afterimmunogold labelling. Routine histological specimens with a thickness ofmore then one micron cause complex reflections. Thinner sections are notgenerally cut for routine histological applications because they do not absorbsufficient light to produce satisfactory image contrast in transmitted lightmicroscopy. Cornelese-ten Velde (20, 21) achieved a significant widening ofapplications in RCM by examining ultrathin (35-70 nm) sections in biologyapplications. Reflection can be very strong in thin specimens if sufficient dif-ference in refractive index exists between the stained cellular components andtheir environment. Cornelese-ten Velde and Prins (22) and Prins et al. (23, 24)made the significant observation that many (immunological) markers like (per-oxidase generated) diaminobenzidine polymer products (DABox), immuno-gold silver, and the alkaline phosphatase staining products show strongreflectance in thin layers. Due to the strong reflection of most immunologicalmarkers they can be simultaneously visualized with most conventional histo-chemical stains. This often allows good morphological localization. Recentlymulticolour RCM detection was developed for in situ hybridization studieswith multiple immune markers (25). For some chemical substances (for ex-ample biomaterials) introduced into cells no histochemical stains are yetavailable. RCM can, however, sometimes visualize such substances if theyhave a refractive index that differs sufficiently from that of their immediateenvironment in the cell.

1.3 Review articlesWestphal (26) discussed the fundamentals of image formation in classicalreflection microscopy for biological applications. Early developments ofRCM are reviewed by Pluta in his handbook 'Advanced light microscopy' (8).Verschueren (16) reviewed reflected-light microscopy in studies on live cells.An extensive analysis of RCM methodology and image formation has beenpublished by Cornelese-ten Velde (20), and combined with applications ofRCM in a thesis (21). More immunocytochemical applications using ultrathinsections were made by Prins et al. (23, 24). Ploem et al. (27, 48) publishedrecent (1995, 1997) general reviews of RCM.

2. Optical systems for RCM2.1 Early developments in reflected-light microscopyIn conventional reflected-light microscopy, scattered light arises at the upperand further lens surfaces of the objective. This causes glare resulting in areduced image contrast. In transparent biological specimens, light reflects alsofrom the underside of the microscope slide if this slide is not oiled to a con-denser. To reduce glare due to scattered light in reflected-light microscopy of

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opaque objects like coal, Stach (14) designed an aperture diaphragm providedwith a central stop creating an annular aperture. These are inserted in theincident light path at a plane conjugate with the back focal (aperture) plane ofthe objective. Piller (15) introduced the 'antiflex' method for reflected-lightmicroscopy of coal. To that purpose such an 'antiflex' objective is providedwith a quarter wave plate in the front lens of the objective. Linearly polarizedlight is led into a vertical illuminator and is then deflected downwards by asemi-transparent beam-splitter mounted above the objective at 45° to theoptical axis. Scattered light reflected upwards from the lens surfaces isblocked by an analyser inserted above the objective, which is crossed withregard to the polarizer. However, light reflected downwards by the beam-splitter transmits the objective towards the surface of the microscope speci-men and is reflected back into the objective, passing the quarter lambda platetwice. The polarization plane of this light changes 2 X 45° = 90°, and thuslight originating from reflective surfaces in the specimen can pass the analyserand go on towards the eyepieces. Zeiss (Oberkochen and Jena, Germany)manufactured the first 'antiflex' (low power) oil immersion objective foropaque specimens (e.g. coal).

Ploem (9, 10) in collaboration with Leica (11-13), integrated both theseoptical methods—used in coal petrology—into a system for high resolutionreflected-light microscopy in biological applications (Figure 1 a). To this pur-pose, new high power oil immersion objectives provided with quarter lambdaplates in the front lenses (suited for high resolution biological and medicalmicroscopy) were developed. For practical purposes, the optical set-up forRCM was incorporated in an epifluorescence microscope equipped with ahigh pressure mercury lamp for incident illumination. An extra relay lens wasmounted in the incident illumination light path (directly in front of the lamp-house) creating an extra aperture plane conjugate with the back focal (aper-ture) plane of the objective. In this plane, special diaphragms with a centralstop creating an annular aperture (precisely adapted to the aperture of thechosen objective) could be inserted. One of the fluorescence filter blocks ofthe fluorescence epi-illuminator is then replaced by a polarizing block en-abling epipolarization microscopy, which is needed as a part of the RCM opticalsystem. Such a microscope can then be used for both fluorescence, epipolar-ization, and reflection-contrast microscopy. Oil immersion objectives pro-vided with quarter lambda plates in the front lens were made for RCM byLeica (X 40, later X 50 and X 100 oil immersion RCM objectives). Zeissmade an X 63 antiflex oil immersion objective for RCM. These objectives,constructed as non-infinity optics are no longer manufactured.

2.2 New developments in reflection-contrast microscopyRecently (1997, Ploem and Prins, personal communication) have developedanother optical method for RCM. A 'normal' X 100 oil immersion objective

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Figure 1. (a) Schematic representation of light path in RCM using an RC objective with aquarter lambda plate in the front lens, (b) Schematic representation of light path in RCMusing a 'normal' objective and a Wollaston prism above the objective, (c) ZeissAxioskopresearch microscope for incident-light microscopy, (dl Leica DMR research microscopefor incident-light microscopy. (e) RCM diaphragm module with different annular aperturediaphragms inserted in Leica microscope stand, (f) Polarizer block.

(without a quarter lambda plate in its from lens) is used, hut a suitahlc Wollastonprism corresponding to the x 100 objective has to be inserted directly abovethe objective. In practice a Wollaston prism as used for differential inter-ference contrast (DIC) can be used. In principle the Wollaston prism in anepi-illumination set-tip achieves a similar function to that of the quarterlambda plate, i.e. turning the polarization direction of the rays reflected from

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the specimen is such a way that this light can pass the analyser towards theeyepieces. As with using the special RCM objective, polarizer block and acentral stop in the diaphragm module in the the epi-illumination light path arerequired (see Section 2.3.1). In principle (Figure 1b) the light, linearly polar-ized by the polarizer, and reflected towards the specimen by the optically flatreflector (beam-splitter), is split by the Wollaston prism into pairs of raysvertically polarized to each other. The image splitting effect is smaller thenthe resolving power of the objective. After reflection from the surface of thespecimen both part-rays are recombined in the Wollaston prism. They thencan pass the analyser towards the eyepieces. The results with oil immersionobjectives with lower magnifications (X 63, X 40) in combination with theircorresponding Wollaston prisms do not give satisfactory RCM results. SinceRCM is especially directed at maximal optical resolution, a X 100 objective isthe best choice.

With most of the very thin and very flat (plane) specimens selected forRCM, practically no inclined surfaces are present and therefore interferencephenomena due to phase differences in the reflected beams do not dominatethe microscope image. Of particular interest for RCM is the possibility of abetter differentiation between metallic tracers used in immuno marking likegold and silver enhanced gold particles and a cytochemical counterstainingused for morphological orientation in the specimen. In research microscopesthe Wollaston prism is mounted in a turret above the objective, and can beshifted by turning the turret slightly. As a result reflectance of a counterstaincan often be reduced (or enhanced) about twofold. In RCM applicationswhere immunogold and silver enhanced immunogold staining are usedin combination with (non-depolarizing) morphological counterstaining orimmunostaining (peroxidase, phosphatase), the visibility of gold and silverparticles (especially when only a few particles are present) can be significantlyenhanced. Shifting the prism quickly back permits again morphological orien-tation based on the counterstaining. The effect is especially seen with ratherstrong counterstaining. With weaker counterstaining the effect is less marked.

Moving the (DIC) turret to a position for bright-field microscopy (noWollaston prism in the path) the gold and silver enhanced gold particles canbe selectively visualized. There may however be a loss of intensity (of abouttwofold) of the reflectance signal but weakly depolarizing stains such asimmunoperoxidase, immunophosphatase, or morphological counterstainingare not clearly visible. Since the RC central stop remains in the illuminatinglight path the image background remains relatively dark.

2.3 Modern reflection-contrast microscopes2.3.1 Upgrading a fluorescence microscope for RCMIt is quite simple to equip a modern epifluorescence research microscope(Figures 1c and 1d) with the few extra parts needed for RCM:

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(a) An oil immersion RCM objective with a quarter lambda plate in the frontlens; or a 'normal' X 100 oil immersion objective with a correspondingWollaston prism mounted above the objective. Usually a Wollaston diskcorresponding to the X 100 objective (as available for differential inter-ference contrast) is accommodated in a turret in the objective nosepiece.

(b) A diaphragm module for RCM (Figure 1e) to be inserted (Figure Id,arrow) in the incident illuminating light path. This module allows theintroduction of a sliding set of annular aperture diaphragms.

(c) A polarizer block is placed in the epifluorescence illuminator (Figure 1 f ) .When thus equipped for reflection-contrast microscopy, the microscopecan also function for transmitted light (absorption) and fluorescencemicroscopy. These three microscope methods can be used sequentiallyand partly simultaneously for the same specimen (Protocol 1).

(d) A protective filter in front of the lamp is required to protect the polarizer(consult manufacturer's manual), if the microscope is exclusively used forRCM. If the microscope is mainly used for fluorescence microscopy andonly infrequently for RCM such a protective filter will interfere withfluorescence excitation light and should not be mounted.

Protocol 1. Adaptation of a fluorescence microscope forreflection-contrast microscopy

Equipment• Fluorescence incident-light research micro-

scope stand with potential for insertion of aRCM module: Leica DMR research micro-scope for incident-light (fluorescence)microscopy (Figure 1d) or Zeiss Axioskopresearch microscope for incident-light (flu-orescence) microscopy (Figure 1c) (arrowsindicate position of RCM module insertion)

• Lamphouse with a high pressure mercuryor xenon lamp as used in fluorescencemicroscopya

• Protection filter for the polarizer (the polar-izer can be damaged by long exposure tointensive light)

• Polarization block, Leica or Zeiss, containspolarizer, neutral reflecting mirror, analyser

• RCM diaphragm module, Leica (Figure 1e)or Zeiss, adapting sliding sets with centralstops and/or aperture diaphragms to beinserted in the incident light path

Fluorescence illuminator with three to fiveexchangeable filter blocks for epifluores-cence microscopy; one block must be re-placed by the polarization block (Figure 1f)

• RCM oil immersion objective equippedwith a quarter lambda plate in the front lenssystem: Leica n-plan oil immersion x100/1.25 RCM objective (infinity correctedoptics) or Zeiss plan-neofluar oil immersionx 63/1.25 antiflex objective (infinity cor-rected optics)

• Alternatively a 'normal' oil immersionobjective: Leica x 100 PL, fluotar 1.30. Thecorresponding Wollaston prism is oftenmounted in a turret above the objective (asavailable for differential interference con-trast). Lower power oil immersion objec-tives with their coresponding Wollastonprisms do not give satisfactory RCMresults.

MethodNote: be careful to observe manufacturer's safety warnings.1. After removing the lamphousing (Leica 105z or 106z), slot the protection

filter for the polarizer into the light exit window of the lamphousingfrom the front.

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Protocol 1. Continued2. Exchange a filter block in the epifluorescence illuminator with a polar-

ization block.

3. Introduce the RCM module into the space reserved for it in the epi-illumination light path of a research microscope stand with provisionsfor incident illumination (research epifluorescence microscope standsof recent design have such provisions).

4. Introduce the sliding units with the various central and aperture stopsinto the RCM module and adjust the central stop and the aperture stopaccording to the manufacturer's instructions for RCM. Centre the fielddiaphragm.

5. Choose the size of central stop in the RCM module in accordance withthe RCM objective.

6. Centre the incident illumination carefully (see Protocol 2), by using theadjusting screws on the lamphousing (see the manufacturer's manualfor high pressure lamps). Adjusting is easier when a Bertrand lens isused.

aMercury high pressure lamps (150 or 100 W) emit strong emission lines dominating thereflectance by the specimen. Xenon lamps (75 W) emit an almost continuous spectrum andprovide 'true' colours.

2.3.2 Light sourcesIn epifluorescence microscopy high pressure mercury and xenon lamps areused and these are also suitable for RCM. In fluorescence microscopy, thelight source has to provide light in the wavelength range absorbed by thefluorochrome. The wavelength of the emitted fluorescence light differs fromthe wavelengths of the absorbed light. In RCM, however, light upon reflectionat the specimen does not change its wavelength. High pressure mercury lampshave a spectrum with the emitted energy concentrated into peaks. Thesestrong mercury lines at, for example 365, 405, 436, 546, and 578 nm, dominatethe reflection by the specimen. Xenon lamps emit a more continuous spec-trum. With this light source the colours reflected by the specimen in RCM arethen not dominated by strong emission peaks. Therefore, they more truly rep-resent the typical spectral reflecting properties of a particular histochemicalstain (see Section 3.1.3). This is especially important if multiple immunohisto-chemical markers are used in the same specimen. They can only by recognizedby differences in the colour they reflect. For specimens stained with multiplestains, a xenon lamp can be of advantage. If only one marker is used for thestaining of a specimen, the mercury emission peaks do not cause a problem.Often the great intensities of such emission peaks result in a stronger reflectionsignal. Most epifluorescence microscopes are usually equipped with highpressure mercury lamps. When such microscopes are upgraded for RCM, one

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usually continues to use the mercury lamp. The microscope can then beeffectively used for both fluorescence and reflection-contrast microscopy.

2.3.3 RCM module for the insertion of central aperture stopsModern research microscope stands for reflected-light microscopy allow theinsertion of an RCM module in the incident illumination light path. The RCMmodule is inserted at an (aperture) plane, conjugate with the back focal(aperture) plane of the objective (Figure 1e).

The Leica RCM module allows the insertion of two sliding holders close toeach other. One with central stops of various sizes and one with different(aperture) diaphragms. Depending on the aperture of each type of objective,a certain size of central stop and aperture must be inserted, to obtain anoptimal image contrast. The aperture diaphragm determines the filling of theobjective aperture (back focal plane of the objective) and thus the angle of thecone of light projected by the objective onto the specimen (28). With bio-logical objects, which are generally quite transparent, most of the incidentlight traverses the slide. It then reflects at the lower side of the glass micro-scope slide, where a large change in refractive index occurs (if no condenser isoiled to the slide). Central stops reduce this unwanted scattered light con-siderably. A detailed discussion of optical conditions for reducing unwantedscattered light in reflection-contrast microscopy is given by Cornelese-tenVelde et al. (29).

Protocol 2. Alignment of the lamp and annular aperturediaphragms in RCM module

Note that exact alignment of the lamp and the central stop are critical forRCM.

1. Centre the lamp (XBO-75) or (HBO-100/150) according to the manu-facturer's fluorescence microscope manual.

2. The projected image of the central stop must be centred in the middleof the image of the arc of the lamp using the Bertrand lens of theresearch microscope.

3. The image contrast of the specimen can be optimized by adjustmentof the collector of the lamphousing.

4. Always realign the arc and the central stop when the lamp is replaced.

2.3.4 Polarizer block in fluorescence epi-illuminatorMost fluorescence microscopes are equipped with an epi-illuminator contain-ing four or more exchangeable filter blocks (cubes). Each block contains anexcitation filter, a dichroic mirror, and a barrier filter for excitation with acertain wavelength for fluorescence microscopy. Blocks for excitation with

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ultraviolet light, blue, or green excitation light are most frequently used. Afourth position may contain a filter block for excitation with violet light or isleft empty to facilitate transmitted-light microscopy. In this position of theilluminator, a polarizing block containing a polarizer, a semi-transparent beam-splitter, and an analyser, can be inserted. With this choice of blocks the micro-scope can be used for either fluorescence or reflection-contrast microscopy ofthe same specimen. (The protection filter for the polarization block absorbssome excitation light wavelengths needed for fluorescence microscopy andmust be removed from the lamphousing. For short periods, however, thepolarization block would not be damaged and could be used unprotected bythis filter.)

2.3.5 Wollaston prism in a DIC turret above the objectiveFor RCM with 'normal' objectives without a quarter lambda plate in the frontlens (or Wollaston prism in the objective) a Wollaston prism, correspondingto the oil immersion objective chosen must be turned in the light path for ver-tical illumination. An aperture diaphragm with a central stop must be insertedin the RCM diaphragm module in the incident illuminating light path, corre-sponding to the aperture of the objective (see Section 2.3.3). The polarizerblock in the vertical illuminator must be turned into its position in the epi-illumination light path. Shifting the Wollaston prism by slightly turning theDIC turret will enable some differentiation between depolarizing and non-polarizing stains (see Section 2.2).

2.3.6 RCM oil immersion objectivesWhen using RCM oil immersion an aperture diaphragm with a central stopmust be inserted in the RCM diaphragms module in the incident illuminatinglight path, corresponding to the aperture of the objective (see Section 2.3.3).A rotating collar around the objective allows the turning of the quarterlambda plate in the front lens of the objective. For RCM, the quarter lambdaplate should be rotated to maximize the reflection from the specimen, atwhich position the polarization angle of the reflected light is at its optimumfor passing the analyser, and RCM is performed. Epipolarization microscopyis obtained when the quarter lambda plate is set to minimal reflectance.

Protocol 3. Focusing on small and thin specimens on microscopeslides

Note: because the specimens selected for RCM are often thin and small,focusing in RCM is a very delicate procedure. An additional difficulty is thatall areas outside the specimen boundaries are observed as dark. For thisreason the instructions below should be followed step for step. Markingthe site of the specimen is necessary. The use of a low power dry objective(X 2.5 magnification) for locating the specimen is essential for easy RCM.

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Equipment• Microscope stand (Leica or Zeiss) with • A marking pen with dark permanent ink

accepts the appropriate central stops andaperture diaphragms for RCM. On the re-volving nosepiece an RCM objective and avery low power dry objective (for examplex 2.5) should be available.

Method

1. Circle marking with a pen closely around the specimen on the non-specimen side of the slide.

2. Put a drop of immersion oil on the marked area of the specimen.

3. Mount the microscope slide with the specimen on the stage.

4. Swing the low power objective into observing position (keep free ofthe immersion oil on the specimen).

5. Search for the marking made on the lower side of the microscopeslide and focus on this marking. The field diaphragm should be simul-taneously visible in the field of view. If you cannot see it clearenough, move the handle of the field diaphragm in the RCM moduleup and down till you notice the field diaphragm.

6. Adjust the field diaphragm in the RCM module to about 75% of themicroscope field by moving its handle.

7. Lower the microscope stage quite a bit by using the coarse stagemovement.

8. Swing in the RCM oil immersion objective into observing positionwithout, however, touching the immersion oil on the specimen (sinceno coverglass is used the objective might damage the specimen byswinging in at too low a level).

9. Raise the stage until the RCM objective just dips into the immersionoil (look from the side of the microscope stage).

10. Move the objective further into the immersion oil with the fine focuscontrol (but avoid the spring-loaded front lens of the RCM objectivebeing pressed inwards). Carefully focus, through the eyepieces ontothe projected image of the field diaphragm (narrowed before to a sizeof 75%). If no image of the field diaphragm is observed, lower themicroscope stage (but without losing contact with the immersion oil),using the fine focus controls until an image of the field stop isobtained.

11. After obtaining an image of the field diaphragm, stop focusing. Youare now in the plane of the specimen. If step 1 of this protocol is car-ried out properly, you are within the marked area of the slide andshould find the specimen by moving the stage slowly in variousdirections.

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12. After microscopic examination the slides should be removed andstored vertically in a microscope slide box, to allow the oil to drainaway from the slide. (Prevent dust collecting on the slides since thismight strongly disturb the RCM examination of the specimen on thenext occasion.)

13. For microscopic re-examination of stored slides, first remove the im-mersion oil still remaining on the specimen with 100% ethanol and letdry. Bring a fresh drop of immersion oil on the specimen before RCM.

3. Image formation3.1 General3.1.1 Reflection of light wavesFor a detailed discussion of image formation in reflection-contrast micro-scopy, the reader should consult Cornelese-ten Velde (20). The reflection oflight at an interface with a given refractive index n and an absorption co-efficient k is described by Fresnel equations. The physical properties n and kof stains can be influenced by the pH, the salt concentration, their conforma-tion (crystalline or amorphous precipitates), and by chemical binding or elec-trostatic forces that may occur during the preparation. In image formation of(stained) microscopic structures in reflection microscopy, three main types ofoptical phenomena and their combination can be observed:

(a) Interference of reflected light waves due to the presence of two or morereflecting interfaces.

(b) Selective reflection of certain wavelengths by a substance, due to its highrefractive index for certain wavelengths. Both interference of reflectedlight (only differences in n) and selective reflection (also contribution ofk) may occur in the same specimen.

(c) Depolarization of reflected light from metal surfaces (high value for k).

3.1.2 Interference of reflected light wavesBy reflection at an interface (Figure 2a), a part of the light wave (I0) is re-flected (Ir) and the remainder is refracted (It) on transmitting the interface (I).

Figure 2. (a) Diagram of multiple reflections in a thin layer system. It illustrates the computa-tion of reflectance. $i = angle of incidence; or = angle of refraction; I0 incident light; lr reflectedlight. (For other symbols see Section 3.1.2.) (b) Schematic drawing of light rays reflecting at acell attached to a glass substrate in an aqueous medium (after Pluta). Rgm: reflection at glassmedium interface. Rgc: reflection of glass-cell contact. (c) RCM image of living cells viewedthrough the coverglass (the objective is beneath the coverglass) to which cells of the kidneytumour cell line SK-RC-52(Gural) are attached. Bar = 10um.

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At the following parallel interface (II) the transmitted (refracted) componentreflects a part (Itr) and transmits (Itt) the rest again. The reflected part of thecomponent ray (Itr) at interface (II) will also reflect and transmit (Itrt) inter-face (I), and so on. The first reflected part (Ir) of the incident wave can interferewith the transmitted part of Itr ( = Itrt) and so on, because they are componentsof the same wave (same frequency), they have a phase difference, and vibratein the same plane (20). Interference of reflected light waves gives informationabout distances between reflecting layers (e.g. glass-cell surface) if they are inthe wavelength range of visible light. Interference of reflected light plays adominant role in the RCM of unstained specimens such as living cells. Inter-ference fringes mostly dominate these images. In thin tissue sections generallyonly a few fringes are visible. In unstained thin sections, when using a xenonlamp a weak colour image can often be observed. This is caused by inter-ference of reflected light waves and depends on the thickness of the section.Interference phenomena can also occur simultaneously with selective reflec-tion, for instance when a dye is present in a layer that produces interferenceof selective reflected light rays. Both account for image formation by theDABox immunocytochemical marker (20) (see Section 3.3). There are twomethods to discriminate between these two phenomena, namely to use inci-dent light of various wavelengths or to use different illuminating numericalapertures (28) (see Section 3.1.3).

3.1.3 Selective reflectionSelective reflection can highlight stains (substances) with special reflectingproperties in the image (mostly more intensively than in transmitted-lightmicroscopy). The resulting enhanced image contrast can simplify the locationof the cytochemical staining. It helps the localization of exceptionally finestructures and very low density in antigen binding sites. Selective reflection oflight can be defined as reflection of certain wavelengths by a substance. It isdue to high absorption and a high refractive index of the substance for thosewavelengths. In reflection microscopy, stained specimens exhibit generally thecomplementary colour of the conventional transmitted-light image, e.g. a bluestained structure in transmitted light (due to the absorbance of red wave-lengths), is seen as red in the reflected light image. This is caused by the reflec-tion of a part of the red incident light at the interface with the stainedstructures. Selective reflection is the more dominant reflection mode withstained specimens (30). Because of the relative complex image formation inreflected-light microscopy, it is always important to investigate the nature ofthe reflected light, to avoid erroneous conclusions such as supposing fluores-cence instead of reflection (30, 31). By changing the wavelength of the inci-dent light and the illuminating numerical aperture (28), Opas and Kalnins(32) could, for example, verify that the stain Coomassie brilliant blue showedmainly selective reflection.

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3.1.4 DepolarizationDepolarization of reflected light can occur with metals. Metal objects in cyto-chemical applications (e.g. silver enhanced immunogold particles) have up tillnow usually been visualized with reflected-light polarization microscopy(epipolarization microscopy). The quarter wave plate of an RCM objectivecan be rotated to minimal achieved brightness of the image. In this situationthe optical configuration of epipolarization microscopy is set. When, however,the quarter wave plate of the RCM objective is turned to maximal brightnessof the image, the optical setting for RCM is obtained. About a twofoldincrease in intensity of the light reflected by the silver enhanced immunogoldparticles is then achieved (29).

3.2 Image formation in RCM of living unstained cellsThe image formation with cells growing on a glass slide has been reviewed byPluta (8). In Figure 2b, a schematic representation is given of a vertical sectionthrough a living cell on a coverglass. When a thin medium layer (e.g. underthe cell) or a thin layer of cytoplasm is present, reflections at both the upper-and underside of these layers occur. When the thickness of the layer is in theorder of the wavelengths of visible light, dark and light bands are seen in themicroscope image. These are caused by interference of reflected light waves.Relatively bright areas are observed at the glass-medium interface where alarge difference in refractive index exists resulting in a strong reflectance oflight. Darker areas are observed at glass-cell surface contacts. A smaller dif-ference in refractive index exists here, causing only a weak reflectance of light(Figure 2c) (2, 3, 6, 7, 33-35). The influence of conical illumination and thesize of the aperture diaphragm has been investigated to estimate the thicknessof cellular structures in living cells (28).

3.3 Image formation in RCM of stained specimensBefore about 1985, mostly non-immunological stains were investigated. Pera(36, 37) studied haematoxylin/eosin stained histological and cytological sec-tions. He noted that when the quarter wave plate of the RCM objective isturned to obtain maximum brightness of the image—as should be done forRCM—an enhanced staining contrast was obtained. Weakly stained cellularstructures, which were only just recognizable in conventional microscopywere clearly visible with RCM. With histochemically stained thick histologicalsections the image information can be quite complex. With RCM of cyto-chemically stained thin specimens often a strong image contrast is obtained.They are often observed against a dark background. Sometimes this maymimic fluorescent images. Van der Ploeg and van Duijn (30, 31) conclude thatsome observations interpreted as fluorescence of stained structures reportedin the literature (38), were due to selective reflection of light. Cornelese-ten

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Velde et al. (20) used a model system to study the image formation of DABox.They showed that the reflection of DABox depends primarily on its thickness,which was estimated at 50-100 nm for sensitive detection of DABox by RCM.From this fact new applications arose: specimens with the stain (DABox) pre-sent in a very thin layer would yield optimal images with RCM. These imageswere obtained from ultrathin epon sections containing DABox and immuno-peroxidase stained Lowicryl sections (20). Pre-embedding is nowadays oftenused for the detection of surface antigens (or extracellular matrix compo-nents) with the PO-DAB as detection method (23). It may be useful to comparethe image contrast obtained with RCM with that obtained by conventionaltransmitted-light microscopy of the same tissue section. The inverted contrastin RCM (bright label against a dark background) cannot however be easilycompared with the transmitted-light image (dark label against a light back-ground). Many microscopes are however linked to a TV camera and connectedto a PC with graphic software for the treatment of digitized microscopeimages. It is then possible to invert (make negative) the RCM image. Whensuch an inverted RCM image is then compared with the original transmitted-light image of the same (semi-thin) tissue section, it becomes clear how largethe gain obtained with RCM is in detecting the immunolabel (Figure 3).Inverting the image is also possible by photographic manipulating, by makingprints from black and white reversal film or colour diapositives.

4. Applications4.1 GeneralCyto- and (immuno)histochemistry are important applications in biology andmedicine. Immunohistochemistry studies using transmitted-light microscopyare sometimes hampered by poor morphological detail and low contrast oflabels in paraffin or cryosections. Bright-field routine sections for histopathol-ogy are generally cut relatively thick (3-6 um) to obtain sufficient image con-trast. When relative thick specimens are used some layers of the specimen are,however, not in focus when a high power objective is used. Thinner sectionswould have been desirable, since practically no out-of-focus layers of the sec-tion would then perturb the image. However, thinner sections lack sufficientimage contrast in transmission microscopy. Although relatively thick specimenscan be examined with confocal microscopy since this optical method elimin-ates out-of-focus information and examines only a thin layer in the specimen.Very thin sections can be examined with reflection-contrast microscopy sincereflection is a surface phenomenon. The microscope images obtained withRCM from ultrathin (30-70 nm) sections have a large image contrast and aretotally in-focus even with high power objectives (Figure 4a). Semi-thin sec-tions (0.25 u.m), however, can provide satisfactory RCM images (Figure 4b).This is important for the routine application of RCM. Most laboratories will

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Figure 3. Localization of immune deposits in nephrology. IgG deposits in the glornerulusof a mouse with graft versus host disease-induced lupus nephritis, visualized by an indi-rect immunoperoxidase-DAB labelling. (a-d) The same 1 um thick section counterstainedwith Light Green seen by (a) bright-field microscopy (BFM), (b) combined transmitted andincident light (BFM/RCM), (c) only RCM, and (d) inverted computer image of (c). The digi-tized image of (c) was only inverted, therefore no colour and brightness enhancementswere performed. Bar - 10 um.

only have standard microtomes available and only a few laboratories will haveultramicrotomes. Most modern motorized microlomes (especially whenequipped with a glass or diamond knife) are very suited to cut semi-thinsections for RCM. Ultrathin sections, however, give superior images.

Like fluorescence microscopy, RCM provides images with a large imagecontrast between the immune label and the tissue background. However,unlike fluorescence microscopy, it also shows tissue background reflections(Figures 5 and 6). Since fluorescent markers can fade, the lifetime of suchspecimens can be limited. With RCM, fading of the cytochemical label is

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(b)

Figure 4. RCM image of (a) ultrathin section and of (b) semi-thin. (a) Ultrathin LR Whitesection of a rat kidney stained with haematoxylin. (b) 0.25 urn thick LR White section of arat kidney stained with haematoxylin. Bar = 10 uM.

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Figure 5. RCM image of binding of PO labelled cMOv 18 on HeLa cells. After developmentof PO with DAB, the cells are embedded in epon (pre-embedded) and ultrathin sectioned.Bar = 10 um.

Figure 6. Localization by RCM of immune deposits in nephrology. IgG deposits inglomerulus of a mouse with graft versus host disease-induced lupus nephritis, visualizedby an indirect immunogold labelling, Ultrathin Lowicryl section mounted on a gelatincoated slide, stained by rabbit anti-mouse IgG, followed by 15 nm colloidal gold-conjugated goat anti-rabbit IgG. Bar = 10 um.

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negligible, so that specimens can be stored and re-examined several times.With a high image contrast and no deterioration of the image by out-of-focusimage formation, RCM of ultrathin sections fulfils the theoretical opticalcriteria of optimal light microscopic resolution,

For some substances (e.g. drugs, particles of various materials as for exam-ple ceramic material) introduced into cells, no cytochemical staining methodshave yet been developed. Detection of such substances by RCM is sometimespossible when such substances have a refractive index that differs sufficientlyfrom their environment in the specimen (Figure 7), In many applications it isimportant to visualize not only, for example, a specific antigenic site in the cellby using an immunolabel, but also to verify the exact morphological site ofsuch labelled objects in relation to other subcellular structures. FortunatelyRCM visualizes all materials in the specimen that have a sufficiently differentrefractive indices. In addition counterstaining can be carried out to obtainf u r t h e r cytochemical or histochemical information. If such a counterstainingwould identify certain other cellular organelles, the precise location of theimmune label would then often be easier. The possibility of using multiplesimultaneous stains in one specimen depends on:

• the differences in the colour of the various stains• the microscopic image contrast obtained

The greater the microscopic image contrast, the easier it is to use the differ-ence in reflectance of a label to distinguish that label from another stain.

Figure 7. RCM image of an degradation of implanted biomaterials. Phagocytosed and de-gradated spheres of injected hydroxyapatite in a giant cell. Ultrathin section. Bar = 10 um.

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RCM of most immunolabels result in strong signals. Many immunocytochem-ical labels can therefore be combined with conventional histo- and cytochem-ical stains. These should, however, be used in a low concentration to give areflectance signal that is just sufficient to observe the cellular structures forwhich the stain is intended. In this way the RCM image looks like an invertedlow magnification EM image. The same cell components are visible, but, oftenwi th a higher image contrast. Interestingly several fluorochromes, such as theDNA/RNA stain. Acridine Orange, can be also used as a counterstain inRCM, since Acridine Orange, in low concentrations and in a thin layer, givessufficient reflection of light. With transmitted-light microscopy, it is not alwayspossible to examine very thin layers and small amounts of biological material,or cells grown on, for example, (non-transparent) filters. Such specimens oftenrequire EM methods for examination. Since RCM is an incident light methodit is possible to examine a great variety of ultrathin sections of specimens (e.g.tissues or cells on a substrate, cultured cells on filters, small biopts).

4.2 Special applications4.2.1 In situ hybridization studiesOne early application of RCM, as mentioned earlier, was the non-radioactivein situ hybridization using pcroxidase-DAB as a label, in genetic studies ofhuman chromosomes. It resulted in the first successful detection of a uniquegene on a human metaphase chromosome using a non-radioactive method(17, 18). RCM is well suited for in sim hybridization studies (Figure 8) and is

Figure 8. Non-radioactive in situ hybridization of mouse metaphase chromosome spreadwith biotinylated pUC 1.77 detected with peroxidase-DAB method. Bar = 10 um.

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Table 1. Comparison of the colour of various enzyme precipitates as revealed by differ-ent types of microscopy after enzyme cytochemical detection of DNA probes in situ (25)

Enzyme reagentsa

APaseAPase

APaseAPasePOPOPOPO

GO

N-ASMX-P + Fast Red TRBCIP + NBT

N-ASMX-P + New FuchsinNBT/BCIP/INTH2O2 + AECH2O2 + chloronaphtholH2O2 + DABH2O2 + TMB

PMS + NBT

Bright-field

RedBlue/purple

RedBrownRedPurpleBrownGreenc,d/purpled

Blue/purple

Microscopy

Reflection-contrast

YellowbOrange/yellowbYellowWhitebYellow"White/yellow"WhitedRedc/yellowc

Yellow"

Fluorescence

Red

Red

a APase = alkaline phosphatase; PO = horseradish peroxidase; GO = glucose oxidase; AEC = aminoethylcarbazole; BCIP = bromochloroindolyl phosphate; DAB = diaminobenzidine; N-ASMX-P = naphthol-ASMX-phosphate; NBT = nitroblue tetrazolium; PMS = phenozine-methosulfate; NBT/BCIP/INT sub-strate, product name (Dako A/S);TMB = tetramethylbenzidine.bFixation of enzyme precipitates in a protein matrix is essential; mounting in immersion oil.c Fixation in a protein matrix is not essential; mounting in immersion oil.d Colour of the reaction product in air dried slides or after mounting in PBS/glycerol.

especially of advantage if the specimens have to be stored to be re-examinedlater. In applications where RCM in situ hybridization methods are applied tocells, nuclear counterstaining may be of help to provide information about thelocalization of the specifically stained structures. Multiple reflecting immunelabels for in situ hybridization studies have been developed by Speel (25)(Table 1).

4.2.2 Rare event detection with RCMThe detection of infrequent objects in medical specimens (e.g. urine andblood) is especially difficult if similarly stained artefacts are also present onthe slide. A careful (and time-consuming) visual inspection is needed of allthese objects which often show only minor differences in image contrast,colour, or signal intensity between specific and non-specific staining. An ex-ample may illustrate this. Microscopic detection of infrequently occurringleptospires in clinical blood samples is difficult. Detection of only a few lepto-spires, using immunofluorescence and transmitted-light microscopy afterimmunoperoxidase staining, is complicated by the fact that some artefactsresemble leptospires. With immunogold silver or PO-DAB staining a verystrong reflectance signal from the leptospires can be seen with RCM (Grave-kamp and Prins; personal communication). Discrimination between lepto-spires and artefacts is easy because of the large image contrast. In addition the

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Figure 9. PO-DAB stained leptospires in a blood-smear. Blood was experimentallyinfected with leptospires (serovar grippotyphosa). Note the characteristic coiling, nor-mally observed with EM. Bar = 10 um. With routine transmitted-light microscopy suchcoiling is difficult to observe and rarely occurring leptospires are difficult to find becauseof a lack of image contrast.

resolution of the microscope image is excellent and characteristic coils of thePO-DAB stained leptospires, (which arc very difficult to distinguish withconventional light microscopy) can be seen in RCM images (Figure 9).

4.2.3 RCM detection of substances with differences in refractiveindex

RCM has been used for example in studies of biomaterials (material for boneimplants). The degradation of calcium phosphate ceramic particles wasstudied in a mouse model system. For RCM ultrathin sections are directly laidon glass slides, strong reflection signals and optimal RCM images can beobtained from biomaterials having sufficiently different refractive indices(Figure 7). This avoids the loss of biomaterial during staining, a problemencountered in processing this material for EM.

4.2.4 Comparison and combination with other microscopicmethods

RCM has been used in comparison and combination with other microscopictechniques such as: differential interference contrast microscopy, high voltageEM, immunofluorescence microscopy, immunoelectron microscopy, scanningEM (39, 40). phase-contrast microscopy, and total internal reflection aqueousfluorescence (41).

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4.2.5 RCM in combination with electron microscopyA methodology in which an overview of a specimen with a resolution close tothe optimal for LM, can be followed with EM examination of the nextsequential section cut from the same tissue block, provides promising possibil-ities. The large image contrast of DABox and immunogold (silver) in RCMmakes an overview and statistical analysis of stained structures in a large partof the specimen relatively easy and efficient. RCM images can be directlycompared with low magnification EM images (22-24). EM can, of course,extend the RCM observations with higher resolutions in smaller selectedareas of the specimen. Such a continuum in observations of the same speci-men with a very large range of resolutions, was up till now not easily possible.This is a practical proposition since often the same pre- and post-embeddingmethods can be used both for light and electron microscopy and can then beprocessed for either RCM or EM (Figure 10).

5. Specimen preparation5.1 GeneralDue to the enhanced detection sensitivity of RCM for many substances, thistechnique is very sensitive to all kinds of contaminants. Contamination by, forexample, dust particles or other unwanted substances must be avoided at allcost (Protocol 3). Microscope slides must therefore be carefully cleaned andtheir surface not touched after they have been cleaned. Precipitates must beremoved from staining solutions. All solutions should therefore be filteredbefore handling the specimens. Coverglasses are not generally used on themicroscope specimens for RCM. After microscopic inspection the specimensshould be stored in such a way that no dust or dirt can accumulate on them.

5.2 ImmunohistochemistryThere are three main types of methods for antigen localization in ultrathinsections by RCM. These methods are common in electron microscopy and theprotocols are (minor) adaptations of these EM methods. Table 2 gives ascheme of the protocols that could be followed for different immunohisto-chemical applications. The choice for a particular method depends on thetype and location of the antigen. If the antigen is unstable (i.e. antigenicityis lost after fixation), ultracryo methods are preferred. When the antigen isstable, its intra- or intercellular localization determines the accessibility of theantibody and by that the choice for pre- or post-embedding methods. Surfaceantigens are best detected by pre-embedding methods, whereas matrix antigens(such as collagen, fibronectin) and intracellular antigens are best detected bypost-embedding methods. Tissue processing for pre-embedding is describedin Protocol 4. Before this procedure is carried out, it is advised to test the

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Figure 10. (a) RCM and (b) EM image of linear deposition of rabbit anti-laminin antibodiesin rat kidney by indirect immunoperoxidase-DAB staining, Ultrathin epon section of pre-embedded tissue, (a) Bar = 10 um. (b) Bar = 5 um.

Table 2. Scheme of protocols: immunohistochemical methods for thin sections in RCM(appropriate number of protocol in brackets)

Pre-embedding

Pre-fixation andirnmunostaining [5]Embedding [5]Ultrathin sectioning [8]Counterstaining [8]RCM [2]

Post-embedding

Fixation and embedding [6]

Ultrathin sectioning [8]Immunostaining (9]Counterstaining [9]RCM [2]

Ultracryo sections

Fixation and freezing [7]

Ultrathin cryosectioning [7]Immunostaining [91or Counterstaining [9!RCM [2]

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antigenicity remaining on 2 um cryosections to determine the concentrationof glutaraldehyde that can be tolerated.

Methoda

1. Fix tissue blocks (c. 4 mm cube) for 3 h at 4°C by immersion in, or per-fusion with, fixative solution.

2. Cut 70 n.m vibratome sections of the tissue blocks and store thesections in wash buffer.

3. Incubate the sections with quench buffer for 60 min at 4°C.

4. Pre-incubate 1% BSA, PBS for 30 min at 4°C.

5. Incubate sections for 16 h with antibody in 1% BSA, PBS at 4°C.

6. Wash five times for 10 min with wash buffer at 4°C.

7. Incubate for 2 h in PO-conjugated antibody in 1% BSA, PBS.

8. Wash five times for 10 min in wash buffer at 4°C.

9. Fix with 1% glutaraldehyde for 10 min at room temperature.

10. Wash three times for 5 min with PBS.

11. Add 10 ul hydrogen peroxide to 10 ml filtered DAB solution.

12. Develop for 30 min in the dark with diaminobenzidine medium.

13. Wash twice with PBS for 10 min.

14. Post-fixate with 1% osmium tetroxide for 30 min.

15. Dehydrate and embed in epon 812 (see Protocol 5). For sectioning seeProtocol 8.

' Modified from the method given in ref. 42, with permission.

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Protocol 4. Tissue processing for pre-embedding

All solutions must by filtered before use with a 0.2 p,m filter (Schleicher &Schuell, FP030/3)

Equipment and reagentsVibratome (Oxford Laboratories, USA)Beem capsules Size OO (Polaron, England)Fixative solutions: 4% paraformaldehyde,0.1-0.2% glutaraldehyde, 0.1 M phosphatebuffer pH 7.31% glutaraldehyde, 0.1 M phosphate bufferpH7.3PBS: 150 mM NaCI, 10 mM phosphate pH 7.3Quench buffer: 0.5 M NH4CI in 0.1 M phos-phate buffer pH 7.3

Wash buffer: 0.1 M phosphate pH 7.3, 3%sucrosePO-conjugated antibody1% bovine serum albumin (BSA) in PBSDiaminobenzidine medium (DAB): freshlyprepared 0.05% diaminobenzidine in 0.05M Tris-HCI pH 61% osmium tetroxide in 0.1 M phosphatebuffer pH 7.330% hydrogen peroxide


Ultracryo methods can be chosen if the antigen is unstable (i.e. antigenicityis lost after fixation). The accessibility for the immunological reagents toreach their targets is excellent. Ultracryo methods are also performed whenantigen location or antigenicity is not precisely known. These methods havenot been developed for conventional light microscopy. RCM, however, offersthis possibility. The excellent RCM image definition of ultrathin cryosections,is comparable to that of ultrathin plastic sections.

Ultracryo and post-embedding methods are quite similar techniques. How-ever, each has different advantages. Handling of resin sections is easier thancryosections, larger sections can be cut, and sequential sectioning is easier.Material embedded in resin blocks and resin sections can be stored for longperiods. Biological material embedded in resin is easy to counterstain.

RCM of ultracryo sections is relatively easy to perform and the whole ultra-cryo procedure is the fastest of the three methods described above. RCM ofultrathin cryosections, in addition, enables the possibility of direct comparisonwith EM.

5.3 Fixation and embeddingParaffin wax embedding and sectioning are not suitable for RCM. Manyroutine biological and medical laboratories, however, increasingly employspecimen embedding in plastics such as GMA, LR White, and Lowicryl. Forsuch laboratories only a relatively small additional effort is needed to makeextra sections for RCM (see Section 5.4). For embedding in plastics similar—but slightly modified techniques—as those required for electron microscopyare used. A selection of the methods used for the preparation of specimensfor RCM is given in Protocols 4-10. For different types of specimens, e.g. cellcultures, biopsies, a variety of fixation methods, pre- and post-embedding inplastic or ultracryo techniques are employed. For further details the reader isreferred to practical handbooks on EM techniques.

Protocol 5 describes fixation and embedding in epon (43) for mainlymorphological studies by RCM. One significant application of RCM of ultra-thin sections is the morphological quality control of cell or tissue cultures.

Protocol 5. Fixation of tissue and embedding in epon for RCM

Equipment and reagents• Razor blade, wax plate, pair of tweezers, • Wash buffer: 0.1 M cacodylate pH 7.3, 3%

Beem capsules, size 3 (Polaron, England) sucrose• Fixative solution: 0.1 M cacodylate buffer • 30%, 50%, 70%, 80%, 90% ethanol in dis-

pH 7.3, 1.5% glutaraldehyde, 1% paraformal- tilled water, and 100% ethanoldehyde (Merck) . DDSA (Fluka, 45345)

. Post-fixation solution: 0.1 M cacodylate . NMA (Fluka, 45347)buffer pH 7.3, 1% osmium tetroxide (AgarUK, R1017) * DMp-30 (Fluka, 45348)

. Propylene oxide (Fluka, 82320) • Oven (temperature range 30°C to 100°C)

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Methoda

1. Prepare mixture of 80 ml epon 812, 50 ml DDSA, and 45 ml NMA. Addto 10 ml of the mixture 0.15 ml DMP-30 (accelerator) and mix.

2. Cut the tissue into small pieces << 2 mm2) with a razor blade.3. Fix for 2 h at 4°C with fixative solution.4. Wash at 4°C overnight with wash buffer.5. Fix for 1 h at room temperature with post-fixation solution.6. Wash three times for 5 min with distilled water.7. Dehydrate with 50%, 70%, 80%, 90% ethanol in distilled water, and

100% ethanol for 30 min each.8. Immerse in propylene oxide for 5 min.9. Infiltrate with epon:propylene oxide (1:1) mixture overnight.

10. Infiltrate with epon:propylene oxide (2:1) mixture for 2 h.11. Embed in Beem capsules in epon and polymerize at 60°C overnight.

* Modified from the method given in ref. 43, with permission.

Protocol 6. Fixation and post-embedding of tissue or cell culturesfor RCM

Equipment and reagents• Razor blade, wax plate, pair of tweezers,

rubber policeman, centrifuge• Conical tip capsules or 1.5 ml Eppendorf

tubes and Beem capsules, size 3 (Polaron,England)

• Ultraviolet polymerization lamp (Poly-sciences Europe, No. 8778)

• 10% gelatin in PBS at 37°C (for embeddingfixed cells)

• Fixative solution: 4% paraformaldehydeand 0.1-0.2% glutaraldehyde in 0.1 M phos-phate buffer pH 7.3

• Aldehyde residue blocking solution: 0.05 Mglycine in PBS

. Wash buffer: 0.1 M phosphate buffer pH 7.3with 3% sucroseLR White (medium 2031) resin (The LondonResin Co. Ltd.)

• Lowicryl: mix 2 g cross-linker mixing with13 g monomer and 0.075 g initiator(Lowicryl K4M kit No. 15923, PolysciencesInc.)

. 30%, 50%, 70%, 80%, 90% ethanol in dis-tilled water, and 100% absolute ethanol

A. For tissuea

1. Cut the tissue into small pieces (< 2 mm cube) with a razor blade.2. Fix tissue for 1 h at 4°C with fixative solution.3. Store tissue for 16 h at 4°C with 2% paraformaldehyde in 0.1 M

phosphate buffer pH 7.3.4. Wash for 2 h at 4°C with wash buffer.5. Dehydrate with 30% ethanol for 30 min at 4°C.6. Dehydrate for 45 min through 50%, 70%, 80%, 90%, and 100% ethanol

at-20°C.

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7. Infiltrate for 45 min with Lowicryl:ethanol (1:1).

8. Infiltrate for 2 h with Lowicryl:ethanol (2:1).

9. Infiltrate in Lowicryl (pure resin).10. Embed in Beem capsules in Lowicryl (pure resin).

11. Irradiate for 24 h with the lamp-to-capsule distance at 30 cm at -20°C.

LR White embedding (alternative to Lowicryl K4M)

1. Follow part A, steps 1-4 as for the tissue treatment.

2. Dehydrate in ethanol 30%, 50%, 70%, 100%, 45 min each, at roomtemperature.

3. Infiltrate in LR White:ethanol (1:1) for 1 h.

4. Infiltrate in pure LR White three times for 1 h each.

5. Embed in Beem or gelatin capsules and polymerize at 4°C for 24 h.

B. For cell cultures

1. Pour off the culture medium and fix cell cultures for 10 min at 4.C.

2. Scrape from bottle with a rubber policeman.

3. Centrifuge (5 min, 100 g) in conical tip capsules, and mix pellet in 10%gelatin, PBS 37°C.

4. Centrifuge (5 min, 260 g) again and cool to 4°C for 10 min.

5. Treat as for tissue (part A, steps 1-11).

aModified from the method given in ref. 44, with permission.

5.4 SectioningBecause for RCM and EM the same specimen preparation method is used, itis possible to obtain subcellular information from the RCM image of a par-ticular site in the ultrathin section (of almost the same tissue structure) byputting a consecutive section on a grid and examining this by EM. Ultracryomicrotomy (Protocol 7) in combination with RCM is a recent procedure foroptimal immune localization (24).

Protocol 7. Fixation and ultracryo freezing and sectioning

Equipment and reagents• Ultracryo microtome (Leica) and glass or • Fixation solution: 4% paraformaldehyde,

dry cryo diamond knife 0.1% glutaraldehyde in 0.1 M phosphate. Dako pen (S 2002) and (3 mm) wire loop 9 buffer pH 7.3

(Figure 77) . Wash buffer: 0.1 M phosphate pH 7.3 with• Aminosilane or gelatin coated glass slides 3% sucrose

(see Protocol 10) . Cryoprotection solution: 2.3 M sucrose in. Hotplate (37 °C) PBS

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A. For tissue (fixation and freezing)a

1. Cut the tissue into small blocks (less than 1 mm3) with a razor blade.

2. Fix tissue for 1 h at 4°C with fixative solution. Or store for longerperiods in 2% paraformaldehyde at 4°C.

3. Infiltrate blocks in cryoprotection solution for 1 h.

4. Freeze in liquid nitrogen on special (cryo) specimen holder.

Sectioning and mounting

5. Cut sections (60-90 nm) on an ultracryo microtome at -120°C with aglass or diamond knife.

6. Transport sections from the knife edge with a wire loop containing adrop of 2.3 M sucrose to a coated slide.

7. Circle the drop of sucrose with a Dako pen, mark the reverse side ofthe slide with water-resistant ink.

B. For cell cultures

1. Fix cell cultures with fixation solution for 10 min at 4°C, remove frombottle with a rubber policeman, embed pellet in 10% gelatin, PBS, andcool for 10 min at 4°C. Fix at 4°C overnight with 4% paraformaldehyde.

2. Cut pellet into small blocks (less than 1 mm cube).

3. Treat further as for tissue (part A, steps 3-6).

a Modified from the method given in ref. 46 with permission.

Figure 11. Picking up resin sections from a water-bath with a wire loop and placing themin a droplet of clean water on a cleaned (coated) slide. Cryosections are picked up with adroplet of sucrose (Protocol 7} and are transferred to a dry slide.

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Semi-thin (0.25 um) sections are well suited for RCM, although ultrathinsections have a superior image definition. Routine plastic embedded tissuecan be sectioned using a glass knife or diamond knife on a routine motorizedmicrotome (Reichert-Jung 2050), that makes cutting of sections down to athickness of about 0.25 um possible. Ultrathin sections made with an ultra-microtome can be brought directly from the diamond or glass knife to a cleanmicroscope slide (see Protocol 10), using a wire loop (Figure 11) (Protocol 8).The sections can then be examined with RCM. Ultrathin cryosections (46)can be brought directly on the microscopic slide and stretch very well withoutsignificant artefacts (Protocol 7). Ultrathin sectioning with an ultracryo micro-tome is now a routine procedure.

Protocol 8. Sectioning of resin embedded tissue

Equipment and reagents• Ultramicrotome or a motorized routine • Coated or clean glass slides (see Protocol

microtome (Reichert-Jung 2050) 70)• Glass or diamond knife • Hotplate• Wire loop 3 mm • Millipore filtered distilled water

Method

1. Cut the sections (250 nm) on a motorized routine microtome, or on anultramicrotome (70 nm sections), using a glass or diamond knife.

2. Collect the sections from the trough, with a wire loop (3 mm) andplace on a water drop on the slide (Figure 11).

3. Dry slide on a hotplate for 45 min at 60°C. (For immunohistochemistrycollect separately on coated slides and dry on a hotplate for 45 min at37°C.)

4. Mark the ultrathin sections on the reverse side of the slide with awater-resistant ink.

5. When pre-embedded specimens are sectioned (see Protocol 4), counter-stain with 0.01% toluidine blue for 5 min, and wash with distilledwater.

6. Store slides in a dust-free slide container.

5.5 Immuno- and histochemical staining for RCMFor a theoretical description of these methods the reader is referred toCornelese and Prins (22), and Prins et al. (23, 24). Immunocytochemistry isdone on resin embedded and cryosections (see Section 5.2 and Protocol 9).

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Protocol 9. Immunomarking of cryo or resin embedded sections

Equipment and reagents

All solutions must by filtered with a 0.2 um filter (Schleicher & Schuell,FP030/3)

• Hotplate • 1% glutaraldehyde in PBS• Incubation buffer: 1% bovine serum albu- • Silver enhancement reagents: developer

min (BSA, Sigma, A-9647), 0.1% gelatin and enhancer solutions (Aurion, The(Merck, 4070), in PBS Netherlands)

• Aldehyde quench buffer: 0.05 M glycine in • Diaminobenzidine medium (DAB): freshlyPBS prepared 0.05% diaminobenzidine in 0.05

. Protein blocking buffer: add 5% serum (in M Tris-HCI pH 6accordance with the second antibody) to • 30% hydrogen peroxideincubation buffer . Light Green (BAH/Gurt, 34204) solution

• Immunogold reagents: IgG conjugated to 1, 0.2% in distilled water5, or 10 nm gold (Aurion, The Netherlands) . Haematoxylin (Merck, 15938) 0.2% in dis-

. DAB, H2O2 solution: add 10 ul H2O2 to 10 ml tilled waterDAB

A. Peroxidase staining1. Carry out all incubations in a moist chamber.2. Quench free aldehyde groups with quench buffer for 15 min.3. Pre-incubate with protein blocking buffer for 5 min.4. Incubate for 1 h at room temperature with serum according to

secondary antibody in a suitable dilution in incubation buffer.5. Wash four times for 5 min with PBS.6. Incubate for 1 h with a peroxidase-conjugated secondary antibody in

a suitable dilution of incubation buffer.7. Wash four times for 5 min with PBS.8. Fix for 5 min with 1% glutaraldehyde in PBS.9. Wash four times for 5 min with PBS.

10. Develop for 10 min in the dark with DAB, H2O2 (see Protocol 4).11. Counterstain PO-DAB for 5 min with Light Green.12. Wash for 1 min with distilled water.13. Dry for 15 min at 60°C on a hotplate.

B. Immunogold staining with silver enhancement1. Follow part A, steps 1-9 (peroxidase staining procedure).2. Develop immunogold—after a few wash steps with distilled water—for

5-10 min with silver enhancer (mix developer and enhancer 1:1 justprior to incubation), and wash five times for 3 min with distilled water.

3. Counterstain IGSS with Light Green or haematoxylin for 5 min.4. Follow part A, steps 12 and 13 (peroxidase staining procedure).

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The immunocytochemical staining efficiency of resin embedded sections(since only their surface is accessible for antibodies) could be somewhat lesssatisfactory than with cryosections. When antigenicity (45) is diminished bythe light fixation with paraformaldehyde, the cryo substitution method de-scribed by Edelmann (47) has considerable potential. In this method freeze-drying and infiltration in Lowicryl in a cryosorption freeze-dryer are used.

If multiple colour immune labelling is done, for example with in situhybridization for chromosomal studies in the same specimen, the differentcoloured end-products may dissolve in immersion oil. If this is likely to occurthen it is advisable to cover the specimen with a thin layer of protein fixativesolution (25) (see Section 5.6).

5.6 Mounting and examining sections on microscope slidesIn immuno applications the cleaned glass slides must be coated (commerciallyavailable coated slides can be used) (Protocol 10). To avoid introducing extrareflecting surfaces, specimens on microscope slides should generally not becovered with coverglasses. If it is necessary to mount the specimen in anembedding medium, this must have a refractive index close to that of glassand immersion oil (n = 1.518). Immersion oil is brought directly onto thespecimen. Again it is sometimes useful to smear a small amount of BSA fixa-tive solution (25) over the slides (to create a thin protective protein layer).This prevents the coloured immuno reaction products dissolving in theimmersion oil. The RCM oil immersion objective is directly dipped into theimmersion oil (Protocol 3).

Protocol 10. Glass slide cleaning and coating

Equipment and reagents• Glass slides ( Menzel-Glaser, Euroslides, • Aminosilane, 2% aminopropylethoxylane

Art. No. 102) (Sigma) in acetone• Glass slides with adhesive coating (Star- • Gelatin, chrome alum: dissolve 0.45% (w/v)

Frost Adhasiv-Objekttrager, Knittel-Glaser, gelatin in distilled water (500 ml), filter, andGermany) add 19.5 ml of a 4% chrome alum solution

. Acetone (PA quality) in distilled water• 96% ethanol

A. For morphological studies

1. Clean slides by washing them for 1 min with 96% ethanol and 1 minwith acetone.

2. Wipe dry with tissue.

B. For immunostaining

1. For coating of slides with aminosilane wash twice for 10 min with ace-tone and coat with aminosilane overnight.

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2. Wash twice for 10 min with acetone, wash for 5 min in distilled water,and allow to air dry.

3. Alternatively use glass slides with adhesive coating (commericallyavailable).

6. SummaryThe few optical parts that are needed for RCM can be easily installed on modernfluorescence research microscopes. Reflection microscopy can, in contrast toabsorption microscopy, visualize very thin tissue sections. All of such a thinlayer of tissue is in-focus even when an objective of high NA is used. Withvery thin specimens the microscope image is not disturbed by pre-focal andpost-focal optical information, as seen with specimens of routine thickness.

Most immunomarkers like peroxidase, alkaline phosphatase, gold, andsilver enhanced gold, show a strong reflectance with RCM, resulting in a largeimage contrast. The detail that can be observed with light microscopy isdependent of both lateral resolution and image contrast. It is therefore notsurprising that RCM provides images with a quality close to the optimalpossibilities of light microscopy (48).

The large image contrast obtained with RCM in immunostaining permitsthe use in the same specimen of classical cytochemical and histochemical(counter)stains. Most of such stains show a weaker reflectance then theimmunomarker stains mentioned above, so that a simultaneous observationof both type of stains is possible. This can be very useful for fine morphologi-cal or further cytochemical orientation in cells and tissues.

The combination of RCM and EM (including ultracryo sections) in examiningthe same specimen is an interesting new possibility. The large image contrastof DABox and immunogold (silver) in RCM makes an overview and statisticalanalysis of stained structures in a large part of the specimen relatively easyand efficient. EM can, however, extend the RCM observations with higherresolutions in smaller selected areas of the specimen. Such a continuum inobservations of the same specimen with a very large range of resolutions, wasup till now not easily possible.

Another possibility of RCM is the study of substances (for example ceramicparticles) in tissues, for which no specific stains are available. Such substancescan sometimes be visualized only on the basis of a relatively small differenceof refractive index in comparison with the tissue in which they are located.

Another interesting possibility of RCM is the study of living unstained cells.The cell to substrate contacts can often be clearly seen. Such images providean information about the attachment of cells to glass surfaces.

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AcknowledgementsWe thank Mr J. Bonnet, from the Laboratory of Histochemistry and Cyto-chemistry of Leiden University, The Netherlands, for his expert advice onRCM methodology.

References1. Faure-Fremiet, E. (1930). Protoplasma, VI, 525.2. Curtis, A.S.G. (1964). J. Cell Biol, 20, 199.3. Izzard, C.S. and Lochner, L.R. (1976). J. Cell Sci., 21, 129.4. Petran, M., Hadravsky, M., Egger, M.D., and Galambos, R. (1968). J. Opt. Soc.

Am., 58, 66l.5. Boyde, A., Petran, M., and Hadravsky, M. (1983). J. Microsc., 132, 1.6. Abercrombie, M. and Dunn, G.A. (1975). Exp. Cell Res., 92, 57.7. Lochner, L. and Izzard, C.S. (1973). J. Cell Biol., 59, 199a.8. Pluta, M. (1989). Advanced light microscopy: specialized methods, 2, 198. Elsevier,

Amsterdam, Oxford, New York, Tokyo.9. Ploem, J.S. (1975). In Mononuclear phagocytes in immunity (ed. R. van Furth),

p. 405. Blackwell, Oxford.10. Ploem, J.S. (1975). Ann. N. Y. Acad. Sci., 254, 40.11. Patzelt, W.J. (1977). Mikrokosmos, 3, 78.12. Patzelt, W.J. (1977). Leitz-Mitt. Wiss. u. Techn., 7, 141.13. Patzelt, W.J. (1976). Naturwissenschaften, 63, 535.14. Stach, E. (1949). Glueckauf, 85, 117.15. Piller, H. (1959). Zeiss-Werkschrift, 34, 87.16. Verscheuren, H. (1985). J. Cell Sci., 75, 279.17. Landegent, J.E., Jansen in de Wal, N., van Ommen, G. B., Baas, F., de Vijlder,

J. J. M., van Duijn, P., and van der Ploeg, M. (1985). Nature, 317, 175.18. Landegent, J.E., Jansenin de Wal, N., Ploem, J.S., and Ploeg, M.V. (1985). J.

Histochem. Cytochem., 33, 1241.19. Hoefsmit, E.C.M., Korn, C., Blijleven, N., and Ploem, J.S. (1986). J. Microsc., 143,

161.20. Cornelese-ten Velde, I., Bonnet, J., Tanke, H.J., and Ploem, J.S. (1988). Histo-

chemistry, 89, 141.21. Cornelese-ten Velde, I. (1990). Reflection-contrast microscopy as a tool in cyto-

chemistry. Thesis, University of Leiden,The Netherlands. ISBN 90-9003710-1.22. Cornelese-ten Velde, I. and Prins, F.A. (1990). Histochemistry, 94, 61.23. Prins, F.A., van Diemen-Steenvoorde, R., Bonnet, J., and Cornelese ten Velde, I.

(1993). Histochemistry, 99, 417.24. Prins, F.A., Bruijn, J.A., and Heer, E.D. (1996). Kidney Int., 49, 261.25. Speel, E.J.M., Kramps, M., Bonnet, J., Ramaekers, F.C.S., and Hopman, H.H.N.

(1993). Histochemistry, 100, 357.26. Westphal, A. (1963). Einfuehrung in die reflexmikroskopie und die physikalischen

grundlagen mikroskopischer bildenstehung. George Thieme, Stuttgart.27. Ploem, J.S., Cornelese-ten Velde, I., Prins, F.A., and Bonnet, J. (1995). Proc.

RMS, 30, 185.

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Microsc., 159, 1.30. Ploeg, M. and Duijn, P. (1979). Histochemistry, 62, 227.31. Ploeg van der, M. and Duijn van, P. (1979). Cytogenet. Cell Genet., 23, 279.32. Opas, M. and Kalnins, V.I. (1984). J. Microsc., 133, 291.33. Beck, K. and Bereiter-Hahn, J. (1981). Microsc. Acta, 84, 153.34. Gingell, D. (1981). J. Cell Sci., 49, 237.35. Gingell, D. and Todd, I. (1979). Biophys. J., 26, 507.36. Pera, F. (1979). Sci. Tech. Inf., 7, 147.37. Pera, F. (1979). Mikroskopie, 35, 93.38. Relief, A.E. (1978). Cytogenet. Cell Genet., 21, 296.39. Gingell, D. and Vince, S. (1982). J. Cell Sci., 54, 255.40. Koerten, H.K., Ploem, J.S., and Daems, W.T. (1980). Exp. Cell Res., 128, 470.41. Todd, L, Mellor, J.S., and Gingell, D. (1988). /. Cell Sci., 89, 107.42. Kerjaschki, D., Noronha-Blom, L., Sacktor, B., and Farquhar, M. G. (1984). J. Cell

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983.45. Newman, G.R., Jasani, B., and Williams, E.D. (1982). J. Microsc., 127, RP5.46. Tokuyasu, K.T. (1986). J. Microsc., 143, 139.47. Edelmann, L. (1994). Scanning Microsc., 8, 551.48. Ploem, J.S., Cornelese-ten Velde, I., Prins, F.A., Bonnet, J., and de Heer, E.

(1997). Sci. Tech. Inf., XI(4), 98.

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8

HistomorphometryA. J. REYNOLDS

1. IntroductionThe analysis of histological material has traditionally been by comparativemorphological analysis. A section of tissue, suitably processed and stained, isexamined under a light microscope by a skilled microscopist and a diagnosismade by observing the morphology of the tissue and cells. Whilst thisapproach is suitable for the majority of analyses conducted, there is a growingneed for a more quantitative methodology. Visual assessment of histologicalpreparations is poorly reproducible and measurement has several advantagessuch as:

• objectivity

• reproducibility

• the ability to detect changes not immediately apparent to the naked eye (1)

A problem encountered in interpreting a microscopic image is that one seesa flat image of a section cut from a three-dimensional object and the viewer isrequired to visualize the spatial context of the object. We are used to seeingobjects as a three-dimensional projection of that object and we have a naturaltendency to interpret flat sections as projections. An example is that a circularprofile will generally be interpreted as being derived from a sphere but, itcould also have come from a cylinder. Problems can become compounded ifwe want to obtain information about the actual size of spatial objects from astudy of sections cut from the bulk. For instance, if a structure composed ofspherical objects is cut by a section plane it is unlikely that all of the objectswill be sectioned through their full diameter, thus most profiles will havediameters smaller than that of the spheres. To assist the microscopist in theinterpretation of sections, mathematical relationships have been derivedwhich relate the two-dimensional 'image' with the three-dimensional 'whole'.The French geologist Delesse proved in 1874 that the volume density of thecomponents of rock can be estimated by measuring the relative areas of theirprofiles (also called the areal density) on random sections cut through therock. This is one of the fundamentals of measurement and proved for the first

A. ]. Reynolds

time that a random section can be a quantitative representation of thematerial from which it is derived.

The techniques used for the quantitative measurement of objects aregrouped together under the term 'stereology'. Histomorphometry is the quan-titative analysis of histological material to obtain a set of data using stereo-logical techniques. Stereology is defined as:

• a body of mathematical methods relating -• three-dimensional parameters defining the structure to -• two-dimensional measurements obtainable from sections of the structure

(2)

Stereological methods have been widely used in geology and metallurgy formany years but it is a relatively new technique in biology being some 30 yearsold (3); and the widespread acceptance of these techniques is largely due tothe efforts of Weibel and Loud.

The basic parameters of stereology are usually the ratios of two quantitiessuch as the number of objects per volume of tissue and a set of definitions pro-posed by Underwood have now become generally accepted (4). A list of thesedefinitions, their abbreviated symbol, and dimensions are shown in Table 1but it is by no means an exhaustive list.

To make the above list more self-explanatory the reference system (thedenominator of the ratio) is written as a capital subscript, for example the

number o f objects p e r unit area i n a tissue section i s : w h e r e T V i s t h e number o f objects counted i n a n area o f tissue A T Similarly t h e

area fraction of these objects is:

where A is the area of the objects in an area of the tissue ATWhen examining sections cut from a bulk material the area fraction AA is

equal to the volume fraction Vv. But, whilst this is true for a single section if itis large enough, the estimates of either area fraction or volume fraction mustbe based on a representative sample. In practice this means that several sec-tions should be measured and the average or cumulative totals used forreporting.

Stereology thus gives the histologist a means of quantifying images of tissuesections to obtain meaningful data concerning the progression of diseasessuch as carcinoma. For instance; biopsies of tumours can be taken at regularintervals and the mean nuclear volume measured to monitor the progressionof the disease and the effects of treatment on the tumour. Stereology is alsouseful in other branches of pathology such as microbiology and cytology.

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Table 1. Symbols used in stereology

Symbol Definition Dimensions

P Number of pointsPP Number of points on feature per point appliedPL Number of cuts per unit length of test line um-1

PA Number of points per unit test area um-2

Pv Number of points per unittest volume (um-3

L Length of element or test line umLL Lineal fraction, i.e. length of line on a feature per unit length

of test lineLA Length of lineal elements per unit test area um-1

L\/ Length of lineal elements per unit test volume um-2

A Area of feature or test area on micrograph um2

S Surface or interface area um2

AA Area fraction, i.e. area of sectioned feature per unit test areaSv Surface area per unit test volume, e.g. membrane area per um-1

unit cytoplasmic volumeV Volume of 3D features or test volume um3

Vv Volume fraction, i.e. volume of a feature per unit test volume -N Number of featuresNL Number of cuts a feature makes per unit length of test line um-1

/VA Number of profiles of a feature per unit test area, e.g. granule um-2

profiles per (um2 of sectionNV Number of features per unit test volume, e.g. mitochondria (um-3

per um3 of cytoplasmL Mean linear intercept length, i.e. LL/NL UMA Mean profile area, i.e. >AA/NA um2

S Mean surface area, i.e. Sv//Vv um2

V Mean volume, i.e. Vv/Nv um3

D Mean diameter, e.g. of a population of granules umd Mean diameter of a population of profiles umv Mean volume of an individual cell or organelle um3

The modern trend towards computer automated analysis has made theacquisition of statistically significant data relatively fast but there is still aplace for the older manual or computer interactive techniques.

2. Microscopy2.1 Specimen preparationHistological specimens are derived from living tissue which is a dynamic sys-tem and thus prone to rapid changes in morphology, especially when deprivedof nutrient and/or oxygen. To minimize these changes the tissue must bepreserved in as near lifelike condition as possible to enable measurements tobe made on the tissue component in question. While no system has beendeveloped that is absolutely sure not to introduce artefacts, the traditionalmethod for light microscopy has been to chemically fix, dehydrate, and embed

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A. J. Reynolds

the tissue in low melting point paraffin wax. A general protocol of specimenpreparation is described in Protocol 1.

Protocol 1. Preparation of tissue specimens for light microscopy

Equipment and reagents• Automated tissue processor (Hacker) or • 74OP IMS (BDH)

glass beakers . Xylene (BDH). Water-bath . Low melting point wax• 10% formalin in physiological saline

Method

1. 10% formol saline, at room temperature, for 0.5 h.

2. 70% alcohol, at 40°C, for 1 h.

3. 90% alcohol, at 40°C, for 1 h.

4. 74OP IMS, at 40°C, for 1 h.

5. 740P IMS, at 40°C, for 0.75 h.

6. 740P IMS, at 40°C, for 0.75 h.

7. 740P IMS, at 40°C, for 0.75 h.

8. Xylene, at 40°C, for 1 h.

9. Xylene, at 40°C, for 0.75 h.

10. Xylene, at 40°C, for 0.75 h.

11. Wax, at 60°C, for 5.5 h.

The resultant wax impregnated tissue is then placed in a suitable mouldincorporating a unique label for the sample and embedded in fresh wax. Amicrotome is used to cut sections of desired thickness suitable for lightmicroscopy (2-5 u.m) which are then attached to microscope slides.

To generate contrast, the sections are stained with suitable polychromaticdyes and covered with coverslips using an appropriate mounting media. Thenumber of stains available probably number in the hundreds and thereforeonly three will be described which have relevance for this text. The first,haematoxylin and eosin (H&E), is the commonest strain in present use forroutine histological preparation (Protocol 2).

Protocol 2. Haematoxylin and eosin stain (H&E)

Reagents• Xylene (BDH) • Eosin stain, 1% aqueous solution. 740P IMS (BDH) . Concentrated hydrochloric acid (BDH)• Harris' haematoxylin staina

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Method

1. Take sections to water:

(a) Dewax in xylene for 3-5 min.

(b) 740P for 10 sec.(c) 90% alcohol for 10 sec.

(d) 70% alcohol for 10 sec.

(e) Running tap-water for 10 sec.

2. Stain with Harris' haematoxylin for 5 min.

3. Wash well with running water.

4. Differentiate with 1% acid alcohol.

5. Wash and blue in running tap-water for 5 min.

6. Stain in 1% eosin for 5 min.

7. Wash in tap-water for 5 min.

8. Dehydrate in graded alcohol:

(a) 70% alcohol for 10 sec.

(b) 90% alcohol for 10 sec.

(c) 740P, twice, for 10 sec.

9. Clear in at least two changes of xylene.

10. Mount in suitable mounting medium.

aThe stain, as bought from the manufacturer, is a poor dyeing agent; to obtain purposefulresults it needs to be converted to its oxidation product haematein. This is achieved by the useof an oxidizing agent such as mercuric II oxide (HgO). Before conversion, haematoxylin isadded to an aqueous solution of alum (usually the potassium salt—aluminium potassiumsulfate). This is the mordanting step, where aluminium ions combine with the haematoxylindye; the resultant aluminium-haematein complex then stains via the metal ion AI2+.

The second stain is a silver stain to highlight nucleolar organizing regions(NORs) which are thought to represent the level of cell proliferation (5).NORs are segments of chromosomes encoded for ribosomal nucleic acid(rRNA), and are present in specific loops of deoxyribose nucleic acid (DNA)which project into the nucleoli where they can be seen by electron microscopyas ill-defined pale staining regions within the more electron dense regions.Some of the NORs can be identified in histological sections by the use of asilver nitrate method which demonstrates an acidic protein with which someof the sites are associated.

It is important to realize that these silver staining NOR associated protein(AgNOR) sites represent only some of the nucleolar organizer regions in eachnucleolus. Furthermore, the spot-like silver reactions seen within the nucleoliin paraffin sections may each represent more than one AgNOR since theytend to be closely aggregated in the nucleoli of normal or benign cells (6).

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This is only one of a number of AgNOR stains; Chapter 4 in this volume listssome alternative stains.

Protocol 3. Silver stain for nucleolar organizer regions (AgNORs)(7)

Reagents• Solution A (50% silver nitrate): 50 g AgNO3 • Working solution: solution A (2 parts): solu-

(BDH) in 100 ml distilled water tion B (1 part), mix immediately before use• Solution B (gelatin solution): 2 g gelatin

(BDH), in 1 ml formic acid (BDH), 100 mldistilled water

Method

1. Take sections to water:

(a) Dewax in xylene for 3-5 min.(b) 740Pfor10sec.(c) 70% alcohol for 10 sec.(d) 90% alcohol for 10 sec.(e) Running tap-water for 10 sec.

2. Rinse sections in distilled water for 2 min.

3. Incubate in freshly prepared working solution for 45 min at roomtemperature.

4. Wash in distilled water for 1 min.

5. Dehydrate in graded alcohol:

(a) 70% alcohol for 10 sec.(b) 90% alcohol for 10 sec.(c) 74OP twice for 10 sec.

6. Clear at least two changes of xylene.

7. Mount in suitable mounting medium.

8. AgNOR sites are intranuclear black dots. The background is paleyellow.

Notes:

(a) Sections may be counterstained with Neutral Red or carmalum. Heavycounterstaining may obscure AgNORs.

(b) Sections may be toned in 1% gold chloride.(c) Working solution deteriorates rapidly on standing.

The third is a general stain called immunoperoxidase and is used, with thesuitable antibody, for the staining of antigen/antibody sites. This technique is

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becoming increasingly popular to show the location of a specific proteinwithin a cell or extracellular matrix such as connective tissue. Some proteinsare specific to particular cell types and antibodies can be used as cell typemarkers. For instance there is a form of actin that marks smooth muscle cells,and the CD3 molecule marks T lymphocytes. The sites of protein stain brownand a counterstain is used to highlight nuclei. Chapter 5 in this volume gives awider description of methods for immunostaining.

Protocol 4. General immunoperoxidase method

Reagents• Dako pen grease-type pencil• Trypsin (Sigma): 0.05 g trypsin, 50 ml 0.1%

calcium chloride (BDH)« Tris-buffered saline: 30.285 g Tris, 16.875 g

NaCI (BDH), 187.5 ml 1 M HCI, distilledwater to 5 litres, adjust pH to 7.6, 0.5 mlTweena

• TBS/0.1% bovine serum albumin (BSA)/0.1% sodium azide: 500 ml Tris buffer,0.05 g BSA, 0.05 g sodium azide (BDH)b

• Swine serum: 5 ml normal swine serum,20 ml Tris buffer pH 7.6

• Diaminobenzidine tetrahydrochloride (DAB):50 ml Tris buffer, two DAB tabletsc

• Copper sulfate (BDH): 4 g copper sulfate,7.2 g sodium chloride, distilled water to1 litre

• Primary and secondary antibody kit (Dako)• Streptavidin (Dako)• Horseradish peroxidase (Dako)

Method

1. Take sections to alcohol (see Protocol 2, step 8).

2. Ring sections with Dako pen. This is to restrict the antibody stainingto the section only.

3. Take sections to water (see Protocol 2, step 1).

4. If trypsin is required, place sections in Tris buffer pH 7.6, for 5 min,then trypsin in calcium chloride 37°C, pH 7.8, for the required timeaccording to the antibody used.

5. Rinse in distilled water

6. Block endogenous peroxide in 3% hydrogen peroxide for 5 min.

7. Rinse in buffer.

8. Immerse in swine serum for 10 min.

9. Drain off swine serum.

10. Incubate with primary antibody diluted in TBS+0.1% BSA and 0.1%sodium azide for 30 min at room temperature.

11. Incubate negatives in TBS/BSA/azide for 30 min at room temperature.

12. Rinse briefly in TBS for 1-2 min.

13. Incubate with secondary antibody diluted in TBS for 30 min atroom temperature; poly-biotinylated goat anti-rabbit 1:500, mono-biotinylated goat anti-mouse 1:300.


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15. Poly and mono kit. To 5 ml Tris buffer add one drop streptavidinand one drop biotinylated horseradish peroxidase.d Incubate for 30min at room temperature.


17. To 5 ml DAB in TBS add 20 ml of 3% hydrogen peroxidee for 10 min atroom temperature.


19. Copper sulfate solution for 1 min.


21. Counterstain with Harris' haematoxylin (see Protocol 2, steps 2-5).

22. Complete mounting procedures as in Protocol 2, steps 8-10.

23. Antigenic sites stain brown. Nuclei stain blue.

aDo not add Tween if oestrogen antibody receptors are the target antigen.b Store in fridge.c Store in freezer.dLeave to stand for 30 min before use." Filter before use.

2.2 Obtaining an imageAlthough it is outside the scope of this chapter to discuss the principles oflight microscopy, it is essential to obtain good quality images that are in-focusand with enough contrast to allow the measurement of features. The tech-niques described in this chapter are all based on the observation of images innormal transmitted light and the microscopist should ensure that the micro-scope is correctly aligned to obtain the best quality image possible. The firststep in obtaining a good image is Kohler illumination.

Protocol 5. Procedure for obtaining Kohler illumination

Equipment• Light microscope with transmitted light source • Slide with good contrast• Phase telescope

Method

1. Switch on the light source and centre it if the microscope lamp hasadjusters.

2. Place a slide with a tissue section on the microscope stage and obtainan image using a x 10 objective.

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3. Partially close the field iris until it appears in the field of view. Focusthe image of the iris using the sub-stage condenser height adjustmentcontrol.

4. Centre the image of the field iris using the sub-stage condensercentring screws.

5. Open the field iris until its boundary is just outside the field of view.

6. Take out an eyepiece and replace it with a phase telescope focused onthe back focal plane of the objective lens.

7. Close the condenser iris until it is about 70% of the back focal plane ofthe objective lens.

8. Replace the eyepiece.

9. When changing objectives the illumination will need to be rechecked.

A more detailed description of setting up the microscope with Kohler illu-mination can be found in Chapter 1 of this volume.

2.3 CalibrationIn any quantitative study, the performance of the testing apparatus must beassessed by careful calibration. In the light microscope, the calibration is per-formed using stage micrometers of known dimensions preferably traceable tonational standards.

For direct microscopical observation an eyepiece graticule is used. This isplaced in the microscope in a position where the scale will appear in sharpfocus and superimposed on the image. The graticule normally lies on the eye-piece diaphragm (i.e. in its focal plane, which is coincident with the primaryimage plane of the objective lens) (8). The eyepiece graticule is calibratedusing a stage micrometer in the following manner.

Protocol 6. Calibration of the light microscope

Equipment• Light microscope • Eyepiece graticule• Stage micrometer

Method

1. Set up the microscope to achieve Kohler illumination (Protocol 5).

2. Obtain an image of a contrasty specimen using the objective lensrequired to make measurements.

3. Replace the slide with a stage micrometer and focus onto the scale.

4. Focus the image of the eyepiece graticule by turning the focus ring of

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the eyepiece until the image of the graticule is sharp against the imageof the stage graticule.

5. Replace the eyepiece of the microscope and align the zero mark of theeyepiece graticule with the zero mark on the stage micrometer.

6. Count the number of divisions on the eyepiece graticule between thezero and the next convenient mark on the stage micrometer.

7. Calculate the distance on the stage micrometer and divide by the num-ber of divisions on the eyepiece graticule to obtain the dimensions ofone eyepiece graticule division.

When making observations from photographic images, a micrograph shouldbe taken of the stage micrometer at the magnification used for recording thespecimen image and printed to the same size as the test micrographs. This canthen be used to calibrate the measuring device used. For calibrating com-puter-based image analysis systems the same techniques can be used depend-ing on whether the image capture system (e.g. video camera) is attached tothe microscope or free standing.

Figure 1. Micrograph of a section of renal carcinoma stained with haematoxylin andeosin on which is superimposed a simple line graticule. The graticule has been printed inwhite to make it stand out against the micrograph. Bar = 10 um.

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3. Linear measurements3.1 Intercept measurementAs the name suggests, these measurements are based on the measuring ofstraight lines either directly on an image or on an overlay (graticule) superim-posed on the image. These are the simplest types of measurement, and theapparatus needed may be no more sophisticated than a ruler. One example ofthis type of measurement is the determination of mean nuclear volume,another example is the estimation of the volume fraction (Vv) of the nuclei, inthe determination of prognosis in renal cell carcinoma (9).

The intercept method uses an overlay graticule ruled with parallel lines.The intercept is the point where the line cuts a feature boundary, i.e. nuclearboundary. In the case of nuclei, there will be an entrance and exit interceptpoint and the linear distance between them is measured with a ruler, in thecase of a micrograph, or with an eyepiece graticule when making the measure-ments directly off the image. Figure 1 is a micrograph of renal cell carcinomastained with haematoxylin and eosin (Protocol 2) with a simple graticule ofparallel lines overlaying the image.

A method for measuring the mean nuclear volume is given in Protocol 7.

Protocol 7. Measurement of mean nuclear volume using thepoint intercept method

Equipment• Overlay graticule of known dimensions • Suitable eyepiece graticule• Stage micrometer • Micrograph of known magnification

Method

1. Superimpose on the micrograph a graticule consisting of a series ofequally spaced parallel lines (see Figure 7) or, if measuring directlyfrom the microscope, superimpose the image of the calibrated eye-piece graticule on the image (Protocol 6). The light microscope mustbe aligned to achieve Kohler illumination (Protocol 5).

2. Measure the total length of line (intercept inside each nucleus. If morethan one line intersects a nucleus, measure the length of each inter-cept and add the two together.

3. Raise each intercept length to the power three.

4. Calculate the mean of the cubed intercept lengths.

5. Multiply the mean value by TT/3 to obtain unbiased estimate of meannuclear volume.

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In stereological terms

where V is the mean nuclear volume, L3 is the total length of lines measuredraised to the power three, and N is the number of nuclei measured.

A worked example of the calculation of mean Value (V) and standard devi-ation (SD) for the nuclear volume is shown in example 1.

These parameters can then be readily used for comparison with those ofother samples and for evaluation of statistical significance of differencesbetween these samples.

Example 1. Estimation of mean nuclear volumeA micrograph of a section taken from a renal cell carcinoma was printed to asize representing X 1000 magnification. A simple line graticule with spacingsof 10 mm (Figure 7), was overlaid on to the micrograph during printing so thatthe graticule is outlined in white. This makes it easier to see against the micro-graph. To perform the analysis the total intercept line length in each nucleuswas measured and the data obtained is shown in Table 2.

Table 2. Line length measurement from Figure 1, at the magnification of the micrograph1 mm = 1 um

Nucleus(um3)

1

2

3

456789

1011121314151617

Line

aabccdeef

gghhh

jk

Line len(u-m)

5437754

10365339292

Line length3 (um3)

1256427

34334312564

100027

2161252727

7298

7298

Sum line length (um) 87 Sum line length3 (um3) 3977

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At X 1000 1 mm is equivalent to 1 um. Total line length3 = 3977 um. Num-ber of nuclei measured (N) = 17. Using Equation 3:

Protocol 8. Measurement of volume fraction by the interceptmethod


Method

1. Superimpose on the micrograph a graticule consisting of a series ofequally spaced parallel lines or, if measuring directly from the micro-scope, superimpose the image of the calibrated eyepiece graticule onthe image (Protocol 2). The light microscope must be aligned toachieve Kohler illumination (Protocol 3).

2. Measure the total length of line (intercept) inside each nucleus. If morethan one line intersects a nucleus measure the length of each line andadd the two together.

3. Measure the total length of the lines on the graticule.

4. Express the total length of intercepts within the nuclei (LNuc) as afraction of the total line length on the graticule (LT) i.e. LNUC/LT.

The mean nuclear volume is 245.0 ± 307.7 um3. The results reflect the largevariation in size of the nuclei typically found in carcinoma tissue.

To measure the volume fraction of the nuclei (Vv) a method similar to Pro-tocol 7 can be used except that the total length of the lines needs to be takeninto consideration. A simple protocol for the estimation of Vv is as follows,again using the example of Figure 1.

Again, in stereological terms:

A worked example using the data in Table 2 and a total line intercept lengthof 847 um using Equation 4.

(The value of 847 um is obtained by dividing the total length of lines on thegraticule in millimetres by the micrograph magnification (X 1000).

A. /, Reynolds

Example 2. Estimation of volume fraction

Volume fraction is 10.27%.

3,2 Point countingThis is similar to the previous examples except that a square graticule issuperimposed over the image and the intersections arc used as points fort Vv

measurement. A square lattice is convenient to use although the use of tri-angular and hexagonal lattices have been described (2). The relative merits ofeach type of graticule have been discussed (10) and the triangular lattice wasthought best. F-Iowever, in practice, square lattices are normally chosen forconvenience. Attention must be paid to the spacing of the lattice and also thenumber of points counted by this technique when considering the accuracy ofthe method. The spacing of the lattice (d) is important for two reasons. Thefirst is that it plays a part in the number of points counted; the more pointscounted the greater the accuracy. The second is that the error inherent inpoint counting, when a fixed number of points are counted, depends partly on

Figure 2. Micrograph of a section of renal carcinoma stained with haematoxylin andeosin on which is superimposed a simple grid graticule. The graticule has been printed inwhite to make it stand out against the micrograph. Bar = 10 um.

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the lattice spacing especially when counting objects with an apparent volume(corpuscular). When measuring Vv of a particular type of organelle, the num-ber of test points needed to achieve the desired accuracy is inversely propor-tional to the square root of Vv and ideally each organelle should have nomore than one point (11). In practice this idealized situation is difficult toachieve and compromise spacings are used, spacings of less than 1 cm are notgenerally used. Figure 2 is the same micrograph as shown in Figure 1 exceptthat a square graticule has been superimposed onto it. The volume fraction isestimated by counting the number of test points that fall within a feature anddividing by the total number of test points. In stereological terms:

where PN is the total number of test points that fall within the objects to bemeasured and PT is the total number of test points.

Protocol 9. Measurement of volume fraction by the pointcounting method


Method1. Superimpose on the micrograph during printing, a graticule consisting

of a grid of squares (Figure 2), the graticule will appear white againstthe micrograph making measurement easier. Print an image of thestage graticule at the same magnification to calibrate the micrographgraticule. If measuring directly from the microscope, superimpose theimage of a calibrated eyepiece graticule on the image (see Protocol 6).

2. Count the number of intersections that fall within the nuclei.3. Count the total number of intersections on the graticule.4. Express the total count of intersections within the nuclei (PNUC) as a

fraction of the total intersections on the graticule (PT) i.e. PNuc/PT.

A worked example of volume fraction estimation is shown in example 3.

Example 3. Volume fraction measurement by the point counting methodA micrograph of a section taken from a renal cell carcinoma was overlayedwith a simple square graticule during printing (Figure 2), the graticule willappear white against the micrograph making measurement easier. Print animage of the stage graticule at the same magnification to calibrate the micro-graph graticule and the total points falling in each nucleus was counted.

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The volume fraction is 10.74%.The volume fractions calculated by the intercept and point counting methods

are very close to each other showing that either method could be used. Inpractice, point counting is preferred as it is easier to count the points than tomeasure lines.

3.2.1 AnisotropyThe above paragraphs describe a lattice system that can be used when the tissueis isotropic in the distribution of the items of interest. This is not always suit-able for some tissues in which the organelles are not randomly distributed(anisotropic). Estimations of Vv in these cases when using a regular squarelattice result in large errors especially when the spacing of the lattice corre-sponds to the periodicity in the sample. The problem can be overcome byplacing the lattice obliquely across the tissue pattern, but a better system touse is one where a series of random points are used. A simple graticule can bedevised where a piece of transparent material such as acetate sheet is markedon two adjacent edges with 100 equally spaced points. A table of random pairsof numbers can be used to plot co-ordinates on the sheet which is then placedon the micrograph and the number of co-ordinates which lie on the features ofinterest counted. The area values can be calculated by the expression:

The random array often yields slightly higher statistical errors than theregular array. But, the random array, in addition to the advantages whenstudying anisotropic tissues, can allow more points to be applied to singlemicrographs since it can be applied repeatedly in different alignments withoutgenerating bias. The graticule should however be rotated between each appli-cation (12). Repeated sampling of a single micrograph with a regular arraygraticule is difficult to achieve with any degree of ensuring independencebetween the samples.

3.2.2 Estimation of the number of points to be sampledThe statistical error of measurement by point counting of a feature is a func-tion of the inverse relationship of a number of points applied to the feature,the less points counted the greater the statistical error. In any micrograph thefeatures to be measured, e.g. nuclei, will be of different relative sizes thus each

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The number of points falling within the nuclei was 13 and the total numberof points on the test graticule was 121.

Using Equation 5:

8: Histomorphometry

feature will accumulate a unique number of points. Therefore the precision ofthe technique will be dependent on the smallest feature of interest. To deter-mine the number of points that need to be sampled, a pilot study is performedto give a crude estimate of Vv. A way to determine the number of points is tocalculate the relative standard error (RSE) using the method of Hally (13)from the expression:

Therefore

and

The value of Vv is the crude value calculated from the pilot study. A workedexample of this is as follows. Consider a value of Vv of 0.2 (20%) with adesired RSE of 5%:

However only 20% of the features occupy the total area of the micrographtherefore we must count at least 320/20 X 100 = 1600 points falling on thewhole micrograph to maintain the desired 5% RSE. This means that severalmicrographs or microscope fields of view have to be measured.

4. Automated measurement4.1 Measurement with an interactive computer system

(digitizer tablet)A relatively cheap way of automating the repetitious measurement of featureson a micrograph or directly from the microscope has become available due tothe falling costs of personal computers (PCs), digitizing tablets, and chargecoupled device black and white cameras (CCD). The simplicity of the equip-ment needed coupled with the operators hand-eye-brain co-ordination makethese systems extremely versatile for the measurement of very complex images.A digitizing tablet or bit pad (Figure 3), is an input device in which a stylus orcursor is moved, by hand, over a flat surface. In general, most digitizing tabletsemploy a grid of wires embedded in the pad which carry a series of high fre-quency pulses. They operate on the principle of magnetostriction which is the

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Figure 3. Digitizing tablet and cursor. The micrograph is fixed to the tablet with stickytape to stop it moving.

change in the dimensions of a ferromagnetic body caused by magnetization ordemagnetization, A pen or cursor detects these changes and interprets themas x and y co-ordinates which are then stored in the computer's memory. Theprocedure for measuring objects is to first calibrate the digitizer tablet with ascale of known dimensions, such as a micrograph of a stage micrometerprinted to the same magnification as the test print, and to then draw round thefeatures of interest with the cursor. The device then records the co-ordinatesof the trace and the computer calculates parameters such as area, perimeter,largest and smallest diameters. The advantage of these devices is that theymeasure features with a greater precision than manual point counting tech-niques and that one tracing can yield several parameters. A disadvantage isthat the accuracy of the system is only as good as the tracing skills of the oper-ator, however if the actual area of the objects to be measured is kept above 16mm2 for a circular profile the errors will be of the order of 6% which is accept-able for most applications in biology. Errors will increase if the shape is morecomplex than a circular profile but this can be somewhat compensated for by

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making the image of the feature larger. Of course, the digitizer must be cali-brated using a linear scale of known dimensions.

4.2 Semi-automatic image analysersUntil the advent of fast personal computers with large memories, automatedmeasurement systems relied on a hard wired computer to perform image cap-ture and analysis with a personal computer used as a 'front end' displaying aninterface program to control the system. Figure 4 shows an automated imageanalyser consisting of a video camera, digitizing pad, personal computer, hardwired computer (under the table), and an image display screen. The videocamera can either be fitted with a lens as a free standing image capture device(as illustrated) or be attached to a microscope for direct analysis. The figureshows the Cambridge Instruments Quantimet Q520 image analyser, the left-hand monitor shows the captured image with the features of interest high-lighted as a graphics overlay, and the right-hand monitor shows the softwareinterface displaying columns of measurements. The graphics tablet for imageediting is in the centre of the figure and the PC mouse is shown to the left. Thesoftware is menu-based with, amongst other options, a detection facilitywhich allows the user to determine the range of grey levels measured (seeSection 4.2.1).

The volume fraction of the nuclei shown in Figure 7 was estimated usingthis system. The micrograph was placed under the video camera and the areasof maximum density (nuclei) were delineated using the detection function.This is done by observing the image on the display screen and using a softwareslider controlled by the PC mouse to fill in the areas of interest. The area frac-tion is determined by a software algorithm similar to the intercept methodalready described. In a two-dimensional image such as a micrograph the areafraction is equal to the volume fraction, i.e. AA = Vv. The value of the volumefraction obtained by this method was 10.37%. To automate the analysis ofsimilar samples which avoids repetitious actions a sequence of commands canbe written called a macro (see Section 4.2.4), but this requires a knowledge ofthe programming language used with the system.

Modern systems now almost always include a personal computer runningMicrosoft Windows™ or Windows 95(™), with appropriate internal framegrabbers and video display cards. Figure 5 shows 'a state-of-the-art' imageanalyser consisting of a personal computer, video camera, and output device;the central processing unit (CPU) and image capture boards are housed in aremote box to free-up bench space. The relative low cost of colour camerashave also made it possible to digitize colour images which is of great import-ance when analysing the products of immunostaining. All of the modernimage analysers incorporate a frame grabber attached to the camera so thatthe images can be digitized and imported into the analyser. The file formatsused are normally GIF or bitmap, although most software packages provide

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Figure 4. Automatic image analyser with hard wired computer. The micrograph is shownon a light box with a CCD camera above. The digitizing tablet allows for operator editingof the image. The right-hand monitor shows the software interface and the left-handmonitor shows the image with the binary digitized detected regions overlayed on top.The hard wired computer is under the bench to save desk space.

the ability to import files from numerous other sources. TIFF, which was thestandard image file formal, seems to be going out of style at present. The sizeof the images captured or imported depends on the system used but even arelatively small image of 1024 X 1024 pixels uses approximately 1 Mb of space(one image can be stored on one high density floppy disk). With computerswith small RAM (random access memory) the programs will run very slowly,a minimum of 8 Mb of RAM is normally required but an increase to 16 or 32Mb of RAM will significantly increase the speed of operation. The cost ofRAM memory has significantly decreased over the last year so that buying asystem with a large RAM does not significantly increase the cost of imageanalyser; and this small extra cost is far outweighed by the time benefitaccrued. Similarly, the speed of the CPU is constantly rising, with the newPentiumTM processors running at speeds in excess of 300 MHz, but bear inmind that a computer with a large RAM and slow CPU may in fact run fasterthan a computer with a fast CPU and small RAM especially when dealingwith images. To conserve disk space there are compression programs that willreduce the size of the image by eliminating blank pixels; but compressionalways results in a loss of data so these programs should be used with care.

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Figure 5, PC-based automatic image analyser, A modern image analyser equipped with acolour camera and microscope adapter. No graphics tablet is shown here although onecan he fitted. The computer is placed under the bench to save desk space. Photo courtesyof Imaging Associates.

4.2.1 Signal processing (erosion and dilatation)The image is recorded by a video camera which converts the image in to a par-allel array of linear scans wi th the video signal reflecting the l ight i n t ens i t y ofthe image l i ne by l ine . By sampl ing the i n t e n s i t y of the video signal at discreetpoints along each l ine, the scan is chopped into lines of points. The resul t ingpoints arc called picture points or pixels which carry two pieces of in format ion :

• s p a t i a l posi t ion

• l i g h t i n t e n s i t y or grey level

The cont inuous band of l i g h t i n t e n s i t y from black to whi le is divided i n t o an u m b e r of grey levels, normally 0 ( b l a c k ) to 255 ( w h i t e ) . The computer candisplay on the viewing screen the position of each pixel of a specified range ofgrey levels corresponding to the component of in te res t in the image, whichcan also be superimposed on the ac tua l image of the sample. This discrimi-nated image is cal led a binary image, also called the segmented image, as eachpixel is e i t he r black ( n o n - d i s e r i m m a t e d ) or whi le (d i sc r imina ted) . The size ofeach pixel can be designated by using a grat icule or l i n e of known dimensions

A. J. Reynolds

and thus it is a simple matter of totalling the number of pixels in each featureto give area measurements or ratioing the number of pixels of interest againstthe total number of pixels to give volume fraction. Modern automatedsystems allow a wide range of parameters to be measured or calculated.

The fact that discrimination of image features depends on their grey levelsleads to two problems. The first is that the specimen must be evenly illumin-ated, which is not always easy to achieve with a light microscope or on amicrograph illuminated by an external source; however most image analysershave correction procedures to deal with this. The second problem is that thegrey level is not always an unambiguous criterion of a certain feature, i.e.other features not of interest may have the same grey level. This problem canbe addressed in two ways. First some image analysis systems are fitted witha digitizing tablet from which the operator can either remove pixels, add infeatures, or separate touching features. This operator intervention is an im-portant feature as it allows the use of very powerful instruments on imagesthat would otherwise not be suitable. Secondly is by the use of techniquesknown as erosion and dilatation or a combination of these two.

To perform either operation it is necessary to define the structuring ele-ment, e.g. a square of 3 X 3 pixels known as a kernel, which is stepped acrosseach pixel in the binary image in turn. The kernel does not have to be a squarearray of pixels; octagonal, hexagonal, and other polygonal structures may beused. The 3 x 3 pixel kernel is the smallest in general use but the number ofpixels in the kernel can be increased to 5 X 5 or larger. A diagrammatic repre-sentation of erosion and dilatation is shown in Figure 6.

In erosion, if all of the pixels in the kernel fall on an image element then thecentral pixel of the image is retained. If, on the other hand, one or more pixelsin the kernel falls onto background when the central pixel is on the image thecentral pixel is set to white. This has the effect of removing one layer of pixelsfrom around each object in the binary image, to enlarge holes, and separatetouching objects. Dilatation is the reverse of erosion; the kernel is againstepped across each pixel in the binary image. The centre point of the kernelwill sometimes fall onto background or sometimes onto an image pixel. Whenthe centre point falls onto an image pixel all of the pixels in the kernel that donot overlap an image pixel are added to the image. This has the effect of addingto the edges of the image and filling in holes. Erosion is a useful tool for re-moving noise from an image as the image can be eroded until all of the pixelscontributing to the noise portion on the binary image are removed. If theimage is then dilatated by the same amount the relevant parts of the imagecan be 'grown' back to their original sizes. A cautionary note—if shape is animportant factor and numerous erosion cycles have been performed, theregrown image elements will not retain their original shape. Erosion followedby a dilatation is called opening and is very useful for cleaning-up the edges offeatures. Conversely dilatation followed by erosion is called closing and isuseful for filling-in cracks and holes in the edges of features.

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Figure 6. A diagram to illustrate the principles of erosion and dilatation, together with their sequential use (opening and closing). Twobasic shapes are shown, that on the left representing an object with protrusions whilst that on the right has fissures. The structuring ele-ment used to erode and dilate is a kernel of 3 x 3 pixels. Note that when opening is carried out on the image with protrusions these areremoved, leaving the basic shape whilst the converse operation closing has to be performed on the fissured image to achieve the sameeffect. From ref. 8 with permission.

A. J. Reynolds

Modern image analysers have functions called ultimate erosion and dilata-tion. Ultimate erosion works like normal erosion except that the regions inthe image remain intact but small regions may be eroded to a single pixel.Ultimate dilatation will grow the regions back to their original size but willplace a single layer of pixels around each object so that objects will not touch.

4.2.2 Image enhancementNot all images presented for analysis may be suitable for measurement, thismay be due to insufficient contrast in the image or other factors such as touch-ing features or noise. Image analysis can often be simplified if the brightnessand contrast can be altered, touching features can be separated, and noise re-duced. Image analysers have facilities for enhancing images by independentlyaltering the brightness and contrast values for each pixel in the image. Touchingobjects can be separated by using erosion and dilatation, or by the use of adigitizing tablet, as discussed in the previous section. By far the most significantaddition to the field of image analysis has been the use of filters for removingnoise and enhancing the image. All of the filters used are mathematical algo-rithms that operate on each pixel in the image but take into account the valuesof neighbouring pixels. A good account of how filters operate can be found inthe book by Glasbey and Horgan (14).

4.2.3 Colour imagingThe dramatic drop in prices of colour cameras and the increase in memory ofpersonal computers has made image analysis of colour images feasible formost laboratories. Most colour cameras operate on the RGB system whichtakes separate images of the red, blue, and green portions of the image. ThisRGB signal is used to display the image in its original colours and also tosegment features of interest by their colour. This is a satisfactory situation formost applications but has one drawback. In RGB, it is the ratios of the valuesof the three channels that determines the colour and also the brightness of theimage which makes it difficult to use the colour information for analysis. Analternative method is to use hue, saturation, and intensity (HSI) where theshade of colour is represented by hue, the brightness by intensity, and satura-tion indicates the mixture of pure colour with white light (15). The advantageof this system is that colour becomes independent of brightness which, whenexamining slides for morphometry, means that the image analyser can be set-up to measure the saturation (which is an indication of amount of staining)and the variations in brightness (intensity) can largely be ignored.

4.2.4 Use of macrosWith the advent of fast personal computers it is now possible to have verysophisticated image processing engines running in real time. The advantage ofthis is that the images can be seen on the computer screen and all of theoperands on the image can be displayed in a gallery of 'thumbnail' images so

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that the operator can see what the effect of a certain command has on theimage. However the computers used by these systems have to be l a s t w i t hlarge RAM capacity (see Section 4.2). The image analyser used as an exampletor i l lus t ra t ion is a Kontron KS300 supplied by Imaging Associates of Thame.Other m a n u f a c t u r e r s of image ana lys i s equipment have their own u n i q u e wayof p resen t ing data and image processing. One of the most u s e f u l functions ofmodern image analysers is the a b i l i t y to write and play macros.

A macro is a s t r ing or sequence of commands that operate on an image andcan i n c l u d e all of the operations needed to process t h a t image. They can besaved on disk and recalled w h e n needed which cuts down cons iderably thel i m e t a k e n for repet i t ious measurements on different images. Unless one isf a m i l i a r w i th the macro, or programming language of an image analyser theeasiest way to write a macro is to process the first image when the macrorecorder is ac t iva ted . Evcry key stroke or f u n c t i o n w i l l be l i s t e d by therecorder as a list of commands. If the macro is stored on disc it can be recalledand replayed lor subsequent images. Macros can also be i n t e r a c t i v e if pausecommands arc included in the macro l i s t . When the macro reaches a pausecommand it wi l l slop and wai l for the user to i npu t the appropriate commandb e f o r e resuming.

The results of r u n n i n g a macro that loads ami processes the image of thek i d n e y carcinoma section t h a t has been used t h r o u g h o u t th is text can be seenin Figure 7 and 8. Figure 7 is the original image captured from the microscope

Figure 7. Image of a section of renal cell carcinoma. The nuclei are stained purple. Theimage shown is the processed image where the brightness and contrast have beenstretched using the image analyser. The image was printed using a colour inkjet printer ata resollution of 300 dots per inch (dpi). Bar 10 um.

Figure 8. Final processed image of renal cell carcinoma with the detected nuclei shownin colour. The coloured nuclei have been overlaid as a graphics plain onto the originalmicrograph (Figure 7). The image was printed using a colour inkjet printer at a resolutionof 300 dpi. Bar 10 um.

using a colour camera and frame grabber. The contras t of the image has beenstretched to improve the de l inea t ion of the nuc le i ( p u r p l e ) from the back-u r o u n d cytoplasm (l ight blue) . Figure 8 is the processed image where a tech-nique cal led segmentat ion was used to detect the areas of m a x i m u m density( n u c l e i ) . This was done by se l ec t ing the pixels in the image w h i c h c o n t a i n e dthe dens i ty range of in t e re s t , in t h i s ease the m a x i m u m and min imum densityv a l u e s of the n u c l e i , from a graphical representa t ion of the t o t a l d e n s i t y rangein the image; th is created a b i n a r y image in which the areas of i n t e r e s t ( n u c l e i )were represented as whi te agains t a black background. The b inary imagewas then overlayed onto the or ig inal image as a graphics plane in which thesegmented p ixe l s have been coloured red to make t h e m s tand out . Theimage analyser was cal ibrated w i t h an image of a g r a t i c u l e that had beenacquired u n d e r i d e n t i c a l conditions to t h a t of the o r ig ina l image and the areaof the p ixels selected by the segmenta t ion procedure measured. The resul t s ofthe a n a l y s i s were au toma t i ca l l y stored in a database f o r m a l . At the end of themacro, the con ten ts of the database were pasted to the clipboard and t h e n to aproprietory spreadsheet package. The spreadsheet was used to c a l c u l a t e thevo lume of each nuclei . the average volume, and the s tandard dev ia t ion . "Theva lue obtained was 265.7 = 414.2 um3 which agrees closely wi th the f i g u r e of245.0 + 307.7 um3 ob ta ined by the i n t e r c e p t method described in example 1of t h i s chapter . Careful examina t ion of the images in Figure 7 and Figure 8shows t h a t the image analyser has overcslimated the size of some nuc le i

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and underestimated that of others. This is because the image analyser is notcapable of such fine discrimination of density changes as is possible with thehuman eye. Whilst this at first seems to be a disadvantage, the benefits ofspeed and the fact that they do not tire, compensate for the slight lack of pre-cision. The image analysers remove the possibility of subjectivity that can befound with human operators.

For analysing comparable images all one has to do is replay the appropriatemacro which does the rest of the analysis. Whilst at first, image processingseems to be complicated, a few hours practice and an understanding of theterminology used in the manipulation of images to achieve the desired resultcan pay dividends in time and reproducibility of analysis.

5. HistomorphometryThe study of the numbers, size, and shape factors of tissue components hasbecome an increasingly popular technique in the diagnosis and prognosis ofdisease. A brief computerized survey of the literature between 1993 and 1996revealed several hundred papers where morphometry was one of the maininvestigative tools. Simple apparatus (e.g. a ruler with a transparent overlaygraticule) can be used for the measurement of mean nuclear volume and volumefraction as discussed in Section 3. Automated image analysers whether a digi-tizing pad attached to a computer or a complete modern image analyser onlyspeed up the analysis for these simple measurements. Table 3 shows theresults of analysis by line graticule (Figure 1), square graticule (Figure 2), andsemi-automatic image analyser (Figure 4) of volume fraction measurementsfrom the same micrograph of renal cell carcinoma, and the time taken for themeasurement.

The three values of volume fraction agree well but the image analyser hasthe advantage in speed. Measuring large numbers of micrographs or fields ofview manually is tiring and can lead to errors whereas large amounts of datacan be accumulated with the minimum of fatigue by automated analysis.

The use of a graphics tablet for the measurement of nuclear shape factorshas been described which helps to distinguish between malignant renal carci-noma and benign oncocytoma (16). Fully automated systems really come intotheir own when more complex measurements such as shape factors, perimeter,

Table 3. Comparison of counting techniques

Volume fraction (%) Time taken (min)

Line graticule 10.27 10Square graticule 10.74 3Image analyser 10.37 0.5

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and centre of gravity are required as the computer performs the necessarymathematical algorithms. Nuclear shape factors when combined with thedetermination of DNA content has improved the ability to predict diseaseprogression in many cancers (17-19). Measurement and numbers of nucleolarorganizer regions (NORs) have been adopted as a measure of cell pro-liferation. These regions in the nucleolus are stained with a silver stain (seeProtocol 3) and image analysers are used to count and size, or calculate volumefraction, of the silver stained regions (20-22). Immunoperoxidase reactions(see Protocol 4) are extensively used for the location and counting of specificcell proteins. These specific proteins are unique to certain cells, thus it is a use-ful way of highlighting these cells for automated image analysis. Rather thancount the individual cells that show a positive reaction, it is probably better todetermine the volume fraction of the tissue stained. The reason for this is thatcounting cells by eye is subjective and large variations can be found amongstpeople counting the same slide. Image analysers have difficulty in separatingtouching objects (15) unless operations on the binary image (such as openingand closing) are performed. Thus it is faster and more meaningful to estimatethe percentage tissue stained.

So far this text has been exclusively about the analysis of stained tissue sec-tions but this does not mean that morphometry cannot be applied to otherfields of biological science. In fact, automated image analysis is used for manyother applications where quantitative analysis of size or shape is required, e.g.the routine screening of cytology samples, blood vessel morphometry (23),sperm head shape in veterinary medicine (24, 25), bacterial shape and colonysize in microbiology (26, 27), and trabecula bone patterns for the measure-ment of bone density (28). This list is by no means exhaustive and the tech-niques described can be applied to almost any image with sufficient contrastto distinguish the features of interest from the background.

AcknowledgementsI am grateful for the help given by Mr Martin Caswell and Mrs Emma Nagleof the Histopathology Department of Wycombe General Hospital for theprovision of slides and access to references. I am also indebted to Mr LeslieStump and Mr Sandy Monteith of Imaging Associates for supplying the Kon-tron KS300 image analyser which was of great help in writing the section onautomatic image analysers.

Instrumentation and sources of supplyIt is very difficult to give a comprehensive list of suppliers of image analysisequipment in such a rapidly changing field and I apologize to those that I haveleft out. The addresses of those companies listed below can be found in theappendix.

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Calibration equipmentGraticules Ltd.Agar Scientific.

Image analysersImaging Associates Ltd.Foster Findlay Ltd.Leica Cambridge Ltd.Synoptics Ltd.Data Cell Ltd.Media Cybernetics.Optimas Corporation.Data Translation.

References1. Hamilton, P. W. and Allen, D. C. (1995). J. Pathol., 175, 369.2. Weibel, E. R. (1979). Stereological methods, Vol. 1, p. 1. Practical methods for bio-

logical morphometry. Academic Press.3. Williams, M. A. (1979). Quantitative methods in biology, pratical methods in electron

microscopy (ed. A. M. Glauert). Elsevier, North Holland.4. Underwood, E. E. (1964). Stereologia, 3, 5.5. Smith, R. and Crocker, J. (1998). Histopathology, 12, 113.6. Bancroft, J. D. and Stevens, A. (1990). In Theory and practice of histological tech-

nique. Churchill Livingstone.7. Ploton, D., Menager, M., Jeannesson, P., Himber, G., Pigeon, F., and Adnett, I. J.

(1986). Histochem. J., 18, 5.8. Bradbury, S. J. (1989). In Light microscopy in biology (ed. A. J. Lacey). Oxford

University Press.9. Artacho-Perula, E., Roldan-Villalobos, R., and Martinez-Cuevas, J. F. (1994). J.

Clin. Pathol., 47, 324.10. Frolov, Y. S. and Maling, D. H. (1969). Cartographic J., 6, 21.11. Hilliard, J. E. and Cahn, J. W. (1961). Trans. Am. Inst. Met. Eng., 221, 344.12. Williams, M. A. (1969). In Advances in optical and electron microscopy (ed. R.

Barev and V. E. Coslett), p. 219. Academic Press, London and New York.13. Halley, A. D. (1964). Q. J. Microsc. Sci., 110, 295.14. Glasbey, C. A. and Horgan, G. W. (1995). Image analysis for the biological

sciences, statistics in practice (ed. V. Barnett). John Wiley and Sons.15. Poston, R. (1996). Image processing, December, p. 4.16. Castren, J. P., Kuopio, T., Nurmi, M. J., and Collan, Y. U. (1995). J. Urol., 154,

1302.17. Linder, S., Lindholm, I., Falkmer, U. S., Blasjo, M., Sundelin, P., and Vourosen,

A. (1995). Int. J. Pancreatol., 18, 241.18. Yoshii, Y., Saito, A., and Nose, T. (1995). /. Neuro-oncol., 26, 1.19. Ruizcerda, J. L., Hernandez, M., Gomis, F., Vera, C. D., Kimler, B. F., O'Connor,

J. E., et al. (1996). J. Urol., 155, 459.

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20. Freeman, J., Kellock, D. B., Yu, C. G. W., Crocker, J., Levison, D. A., and Hall,P. A. (1993). J. Clin. Pathol, 46, 446.

21. Derenzini, M., Trere, D., Oliveri, F., David, E., Colombatto, P., Bonino, F., et al.(1993). J. Clin. Pathol., 46, 727.

22. Kossard, S. and Wilkinson, B. (1995). J. Cutaneous Pathol., 22, 132.23. Tipoe, G. L. and White, F. H. (1995). Histol. Histopathol., 10, 589.24. Gravance, C. G., Lewis, K. M., and Casey, P. J. (1995). Theriogenology, 44, 989.25. Gravance, C. G., Lin, I. K. M., Davis, R. O., Hughes, J. P., and Casey, P. J. (1996).

J. Reprod. Fertil., 108, 41.26. Schafer, C. (1992). Image News, 2,16.27. Wilkinson, M. H. F. and Meijer, B. C. (1995). Compter methods and programs in

biomedicine, 47, 35.28. Chappard, E., Legrand, E., Basle, M. F., and Audran, M. (1997). Microsc. Anal.,

62,23.

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Near-field optical microscopyNIEK F. VAN HULST

1. Introduction

1.1 Optical microscopyThis is a chapter on an emerging field in optical microscopy: near-field optics,i.e. the optics of subwavelength-sized structures. It may seem surprising tofind any aspect unexplored within the mature and well-established domain ofoptical microscopy. Indeed optical microscopy has reached an outstandinglyhigh level of perfection through centuries and established itself as the maintool in biological studies through its flexibility, applicability, and multitude ofoptical contrast methods. Yet, a natural limitation had always to be faced: thediffraction limit. It is fundamentally impossible to focus light to an areasmaller than the size of the wavelength. Consequently the spatial resolution inlens-based optical microscopy is limited to about half the wavelength. Thisbarrier, already recognized in the previous century by Abbe, has been astimulus to develop alternative techniques like electron and ion microscopy.Indeed these alternatives have pushed the resolution to the atomic level (intransmission electron microscopy), however generally with the loss of the'optical' advantages, such as: non-invasiveness, non-destructiveness, opera-tion in native environment (in vivo), high resolution spectroscopic contrast,polarization contrast, and time resolution. The optical approach towardssubwavelength resolution naturally involved phase-contrast, however thenear-field domain beyond the diffraction limit, dealing with non-propagatingwaves, has remained until recently beyond the scope of classical opticians.Nowadays in the fields of biology and microelectronics nanometre-sized struc-tures are gaining increasing significance, making the study of the near-fieldoptical properties of 'nano-structures' highly appropriate and stimulatingboth near-field optical instrumentation and theory. Experimental near-fieldoptics has taken off in 1982, directly following the development of scanningtunnelling microscopy in 1981 (1). Though among the first in the expandingvariety of scanning probe methods, near-field optics has been suffering theproblem of probe fabrication for almost a decade. Only gradually, with im-proving efficiency and versatility near-field optics has started to show its latent

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promises of 'optical contrast' at nanometre dimensions (2). The steadily grow-ing number of near-field optical biological applications is indicative of itsfuture importance to light microscopy in biology (3).

1.2 Probe microscopyIn the last decade, scanning probe microscopy (1, 4) has emerged as a usefultool in biological research. Especially, atomic force microscopy (AFM) hasdemonstrated nanometre scale resolution on biological samples at a variety ofconditions (5). The introduction of 'tapping mode' AFM (6) and specificallythe operation in liquids (7, 8) has enabled the imaging of living biologicalmaterial, almost unperturbed in near-physiological conditions (9). Indeedactivity of cells (9, 10) and enzymes (11) has been followed with high spatialand temporal resolution (12). Lately the intrinsic chemical specificity of AFM,as contained in the very nature of the force, is successfully being exploited for(bio)chemical mapping. Thus 'adhesion mode' AFM has been developed (13),where the difference in chemical bond strength between probe and sample isused as a contrast mechanism. By additional coating of the force sensor with adefined molecular monolayer the interaction can be made specific, leading to'molecular' force microscopy (14). However, despite the outstanding verticaland lateral sensitivity and emerging chemical specificity of force sensing,optical detection has remained essential to biological investigation, due to itsconvenience, non-invasiveness, and the extensive variety of contrast mecha-nisms associated with light. In particular, the chemical specificity contained inspectroscopic information is vital for the understanding of many biologicalprocesses. Thus combination of probe microscopy with an optical contrastmechanism has the potential to combine the best of both. This is the domain ofnear-field optical microscopy (15). The feasibility of near-field optics has beenexplored experimentally immediately following the start of probe microscopy,even before AFM, by Pohl et al. (16). Yet among the probe techniques near-field optics has long been considered as an academic peculiarity, especially com-pared to the enormous impact of AFM. Only in recent years through decisivetechnological achievements, such as the development of adiabatic fibre pullingand shear force feedback in 1991 by Betzig et al. (17), near-field scanning opti-cal microscopy (NSOM) has gained recognition as a microscopic techniqueoffering optical contrast beyond the diffraction limit with rapidly expandingapplications in material science, surface chemistry, and indeed biology (2).

1.3 Breaking the diffraction limitThe minimum spot size of a standard or confocal optical microscope is limitedby diffraction to approximately 0.5 \/NA, i.e. ~ 300 nm in the optical regime(18). Using a shorter wavelength, such as ultraviolet light, another factor of twomight be gained. However, both the lens materials and the biological material

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are getting opaque towards far UV wavelengths, rendering both lens fabrica-tion and application problematic. The detection of phase yields a vertical res-olution down to the nanometre scale, but the lateral resolution is still limitedby diffraction. Currently the detection of lateral phase information, in so-called 4rr eon focal microscopy, is an active field of research in an effort toachieve full nanometric resolution in three-dimensions using lens-based opticalmicroscopy (19).

In contrast to far-field optics based on propagating waves, near-field opticsis restrieted to suhwavelength dimensions, i.e. the optics of non-propagatingwaves. As a consequence near-field optics does not (cannot) apply lenses, butis based on an antenna (a nanometre-sized dielectric or metallic structure)interacting on subwavelength scale with the local field of a sample to he ex-amined. The local interaction generally modulates the far-field radiation,which is subsequently collected using conventional optics. The interactionvolume is determined by the size of the antenna, basically independent of theapplied wavelength.

The concept of near-field optical microscopy, with a resolution beyond thediffraction limit is schematically illustrated in Figure 1 for the case of anaperture-type antenna. To the left is sketched the diffraction limited f i n i t ebeam waist (> \/2) of a tightly-focused beam. In the middle the far-field spotsize is geometrically confinement to a size a « \ using metallic screening withan aperture. For practical purposes of operation, the actual configuration ofan aperture-type near-field optical probe is based on a tapered optical fibrewith a metallic coaling for screening off the far-field contribution, as shown onthe right side of Figure 1.

Figure 1. Breaking the diffraction limit Schematic illustration of (left) the diffractionlimited finite beam waist (> \/2) in a tightly-focused beam, (middle) the geometric con-finement to a size a « \ using metallic screening, and (right) the actual configuration of anaperture-type near-field optical probe, based on a tapered fibre with Al coating.

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1.4 Scope of this chapterThis chapter is focused on near-field optical instrumentation and applicationsin biology. First an overview of several near-field optical microscopic arrange-ments will be presented with a discussion on specific advantages and dis-advantages as to resolution, efficiency, versatility, and applicability. Nextnear-field transmission, absorption, polarization, and fluorescence microscopyof various biological surfaces is shown with a lateral optical resolution typi-cally below 100 nm, depending on the arrangement used, but mainly usingaperture-type probes:

(a) Single fluorophores and proteins.(b) Monolayers and J-aggregates, visualizing the orientation of the molecular

dipole and polymer backbone.(c) A virus.(d) Fluorescence of cytoskeletal actin.(e) Chromosomes and fluorescence in situ hybridization (FISH), where the

localized fluorescence allows the identification of specific DNA sequences.

The presented images are all beyond the diffraction limit and generallyaccompanied by the simultaneously acquired topographic force image, en-abling direct comparison of the optical contrast with the sample topographyon nanometre scale. It will be argued that the unique combination of highresolution, specific optical contrast, and ambient operation opens many yetunexplored directions in biological studies.

2. Instrumentation2.1 Probes and distance regulationThe heart of any near-field optical microscope is the near-field optical probe: ananometre-sized antenna that interacts with the sample and transmits the mod-ulated response to the observer, while scanning over the sample surface atnanometre distance. The probe determines the contrast mechanism, the spatialresolution, and the sensitivity. As such the importance of the probe is compara-ble to that of the objective in far-field optical microscopy. Many types of probeshave been fabricated and applied in various configurations. Figure 2 gives a

Figure 2. Types of near-field optical microscopes. Schematic overview of the variousexperimental configurations for near-field optical microscopy. Top: the antenna (or'apertureless') type. A nanometric-sized particle, acting as a source or detector (left), isscanned in close proximity over a sample surface. In practise the particle is a passivemetallic tip (right), excited by far-field illumination, where the local interaction with thesample surface is detected as a modulation in the scattered far-field. Middle: the aper-ture-type near-field scanning optical microscope (a-NSOM). A subwavelength-sized aper-ture is scanned in close proximity to the sample surface. The modulation of the aperture

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transmission caused by optical interaction with the sample is detected in the far-field,either by illuminating (transmission mode) or collecting through the aperture (collectionmode). Bottom: the photon scanning tunnelling microscope (PSTM). An optical field,evanescent perpendicular to the surface and propagating along the sample surface, iscreated by dark-field illumination at an angle beyond the critical angle for total internalreflection. The evanescent wave is locally frustrated by a dielectric probe (a tapered fibreor a micro-fabricated lever structure) and converted into a propagating wave. Thefraction coupled into the probe is detected in the far-field.

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schematic overview of the main experimental configurations encountered inpractical near-field optical microscopy, divided into the antenna (or 'aperture-less')-type, the aperture-type, and the photon tunnelling configuration.

2.2 Antenna-type or 'apertureless' near-field scanningoptical microscopy

Ideally the near-field optical probe is of molecular dimensions, serving as asource or detector, which is scanned in close proximity over the sample sur-face (Figure 2, top). The probe has to be manipulated in a scan pattern overthe surface and addressed to collect the near-field interaction information. Asa result the probe will be rather a macroscopic tip ending in a nanometre-sized apex supporting the source or detector. Fabrication of an optical sourceor detector of nanometre-size dimensions is a challenge in itself. Althoughefforts based on luminescent particles, polaritons, or submicron-sized photo-diodes have been undertaken, none of these has been fully developed. Inpractice the antenna is simply a passive metallic tip, excited in far-field illumin-ation by a tightly-focused spot, and the local interaction with the samplesurface is subsequently detected as a modulation in the scattered far-field.Extreme sensitivity is required to observe the weakly scattered light from thenanometre-sized tip in the presence of light scattered from the sample. Zen-hausern et al. (20) have developed a sensitive interferometric detectionscheme, while vibrating probe and sample, to discriminate the tip-sampleinteraction from the background. Images with resolvable features a small as afew nanometres were obtained with contrast based on dipole-dipole coupling.Similarly Koglin et al. (21) obtained 6 nm resolution, in absorption contrast,by using small gold grains on a sharp glass support. The nanometric lateralresolution, far beyond the diffraction limit (even approaching atomic resolu-tion), is illustrative for the potential of probe methods. However these con-figurations are far from routine, only operational on rather specific samples,and require strong illumination conditions. Biological applications based onmetallic tip interactions are yet to be demonstrated.

2.3 Aperture-type near-field scanning optical microscopyThe most widely accepted and applied configuration is the aperture-typenear-field scanning optical microscope (a-NSOM, Figure 2, middle): a sub-wavelength-sized aperture is scanned over the sample surface at a distance ofa few nanometres. A small fraction of the light passes the aperture and isdetected in the far-field, either by illuminating (transmission mode) or collect-ing through the aperture (collection mode). The close proximity of the aper-ture to the sample surface, with its spatially varying refractive index, opacity,and anisotropy, results in a modulation of the aperture transmission dependingon the aperture position. Additionally the sample topography affects theaperture transmission. Thus images are obtained containing index, absorption,

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polarization, and topographic contrast. Moreover detection of non-resonantlight, such as fluorescence, gives valuable spectroscopic information.

The practical feasibility of near-field optical microscopy was first demons-trated by Pohl et al. (16) in 1982 with an optical super-resolution of 20 nmusing an aperture at the apex of a sharpened quartz rod. Further developmenthas for long been hampered by fabrication problems of efficient apertureprobes. Since 1990 considerable progress has been made with the advent ofefficient and reproducible fibre probes, as introduced by Betzig et al. (17, 22).This near-field aperture probe is fabricated by adiabatic tapering of an opticalfibre using a special purpose fibre puller (Sutter Instrum. P2000) and subse-quent directional coating with ~ 100 nm of aluminium. Thus a 50-100 nmdiameter aperture is created, with surrounding aluminium for screening of thefar-field. The probe has a brightness of 1-10 nW when several mW laser lightis coupled into the fibre, i.e. an efficiency of 10-6 to 10-5, where the inputpower is limited by the thermal damage threshold of the aluminium coating.

The electric field at the probe aperture is highly non-homogeneous due tothe metal coating and the polarization of the light: e.g. the electric field is zeroat the position where the polarization is parallel to the metal coating.Bouwkamp (23) derived theoretical expressions for the electromagnetic fielddistribution behind a small aperture (radius a « \) in a perfectly conductingmetallic screen. Figure 3 shows the electric field intensity (|E|2) profile atseveral distances (\/500, \/100, \/20, and X/4) behind a small aperture witha = X/20, with incident light polarized in the horizontal x direction. At shortdistances (< \/100) the size of the distribution is confined to the dimension ofthe aperture. At larger distances (> X/4) the field decays rapidly and the con-finement diffuses to dimensions rapidly exceeding the size of the aperture.Consequently high resolution imaging can only be expected while operatingin close proximity at a distance of only a few nanometres.

Using the relatively efficient aperture probes first application of near-fieldoptical microscopy to biological and chemical samples has been explored byBetzig et al. (24) and Moers et al (25). Moreover the pulled fibre probeenabled fluorescence detection of a single molecule, an important achieve-ment in the field of molecular physics, first demonstrated by Betzig andChichester (26) and immediately followed by single molecular spectroscopy(27) and single molecular fluorescence lifetime detection (28).

For biological applications it is important to design the near-field micro-scope such that standard object glasses or coverslips can be accommodatedand the sample can be viewed with conventional high magnification optics forlocalization of a specific area of interest. Often the near-field optical micro-scope is based on an inverted microscope with sufficient mechanical stabilityon the nanometre scale (e.g. the Zeiss Axiovert). The inverted configurationleaves sufficient space for the mounting of a near-field optical probe in theimmediate vicinity of the sample surface. The commercial sample table hasto be replaced by a combined mechanical and piezo-electric scan table with

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Figure 3. Electric field intensity profiles IEI2, in x, y, and z direction, in several planes(z = \/500, \/100, \/20, and \/4) behind a small aperture (radius a \/20) in a perfectlyconducting metallic screen, as calculated according to the theory of Bouwkamp (23), withincident light polarized in x direction. The grey scale is normalized for optimum contrastin each plot. Close to the aperture the field is concentrated at the rim due to the abruptdielectric/conductor transition. For increasing distance the field decays rapidly and theconfinement diffuses to dimensions beyond the aperture.

sufficient range for coarse and fine positioning, A high NA objective (0.75 NAdry or 1,4 NA immersion) is used for efficient collection, over a large angularrange, of the light transmitted by the aperture in interaction with the sample.For fluorescence detection a dichrotc mirror and long-pass filter are used,which block the excitation light, identical to far-field methods. In near-fieldoperation the probe source is confocally imaged onto a point detector,mounted at one of the microscope exit ports. For high light levels, > 1 fW, aphotomultiplier tube in combination with a pin-hole (~ 100 um) in the image

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plane is generally sufficient. For low light levels, < 106 photo-counts/sec, a100 (Jim area photon counting avalanche photodiode (EG&G, Electro-Optics)is preferentially used because of the high quantum efficiency (~ 59% at\ = 600 nm) and low dark count level (< 10 photo-counts/sec).

2.4 Photon scanning tunnelling microscopy (PSTM)An alternative arrangement is photon scanning tunnelling microscopy(PSTM, Figure 2, bottom), based on frustration of total internal reflectionwith a sharp uncoated dielectric probe and operated in transmission mode.The optical field is created by dark-field illumination at the inner side of a sup-porting glass substrate at an angle beyond the critical angle for total internalreflection. The resulting field is propagating along the sample surface andnon-propagating (evanescent) perpendicular to the surface. The presence ofa sample at the glass-air interface modulates this field and generates newnear- and far-field components. The perpendicular evanescent wave is locallyfrustrated by a dielectric probe and converted into a propagating wave. Thefraction coupled into the probe by 'optical tunnelling' is detected in the far-field. The probe can be an uncoated tapered fibre or a micro-fabricated struc-ture (29). Micro-fabricated silicon nitride (SiN) probes are commerciallyavailable (Park Scientific Instruments) for conventional AFM applicationsand are suitable high-index probes with 20-50 nm apex and transparency intothe UV. Moreover due to the integrated cantilever the probe can be scannedin close contact with a sample surface with feedback regulation on the forceinteraction. The combined PSTM/AFM yields simultaneously a topographicand a near-field optical image (30).

The operation with uncoated dielectric probes makes PSTM experiment-ally easier than 'aperture' NSOM, occasionally yielding very high lateralresolution, down to 20 nm (29). However PSTM responds strongly to far-field scattering, which limits the method to samples with topography muchsmaller than the wavelength. PSTM images of biological objects are generallydominated by far-field scattering, making it hard to find suitable biologicalapplications.

2.5 Distance regulation: shear force microscopyThe use of a fibre probe in close proximity to the sample surface, typically afew nanometres, requires a highly sensitive distance sensing and regulatingmechanism to avoid the fibre from crashing into the surface. Fortunately withthe development of scanning probe microscopy this problem has been solvedthrough the use of piezo-electric manipulators and sensing of probe-sampleinteraction based on electron tunnelling or atomic forces. In the case of bio-logical applications the sample will generally not be electrically conductive,which eliminates the possibility of electron tunnelling. Force sensing has been

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utilized in the many biological AFM applications. Consequently the methodof choice is to combine near-field optical and force sensing. Force microscopybased on tapered fibres was demonstrated in 1992 by Toledo-Crow et al. (31)and Betzig et al. (32). They attached the fibre probe to a piezo-electric ele-ment, which oscillates the fibre in the lateral direction (parallel to the samplesurface) with an amplitude of about 20-30 nm at its resonance frequency(typically > 10 kHz). The oscillation amplitude is measured with a sensitivityof ~ 1 nm using optical detection, either by interferometry (31) or by project-ing the far-field diffraction pattern on a split detector, where the differencesignal is a measure for the fibre amplitude (32). The oscillation amplitudedecreases on approaching the sample surface due to 'shear' forces betweenprobe and sample. The distance between the aperture probe and the samplesurface can be adjusted between 1-15 nm, with subnanometre accuracy, by afeedback system based on this shear force detection. Simultaneously a topo-graphic 'shear' force image of the surface is obtained, similar to regular AFMoperation.

The optical detection of shear forces has the disadvantage that additionalstray light is brought into the vicinity of the aperture and that accurate align-ment of the detection system with respect to the probe is necessary. An alter-native method is to use the piezo-electric material itself to generate a voltageproportional to the amplitude of the oscillation. Based on this idea, the use ofquartz crystalline tuning forks was developed by Karrai et al. (33). In this case,the end of the fibre is attached to one arm of a tuning fork and the fork isoscillated at resonance (usually 32 kHz). Again on approaching the samplesurface, a decrease of the oscillation amplitude of the tuning fork is observed,which is subsequently used for distance regulation. The tuning fork configura-tion can be realized very compactly and has a vertical sensitivity of ~ 0.1 nm(34).

2.6 ConclusionAperture-type NSOM based on metal coated adiabatically tapered fibres,combined with shear force feedback and operated in transmission mode, hasproven to be the most powerful NSOM arrangement, because of its true local-ization of the optical interaction, its various optical contrast possibilities (fluor-escence, polarization, etc.), and its sensitivity down to the single molecularlevel. The system has been commercially available from TopoMetrix Corp.(35) since 1993, and their 'Lumina' microscope is illustrated in Figure 4. In1996 Nanonics (36) has launched an alternative configuration, utilizing a bentfibre probe. Both systems are based on a commercially available invertedmicroscope, enabling all contrast modes of conventional optical microscopy,such as phase, DIG, reflection, and fluorescence. A sample area of choice canbe examined in further detail with combined near-field optical and forcemicroscopy.

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Figure 4. The 'Lumma' NSOM of TopoMetrix Corp. The system is based on a researchgrade commercial inverted optical microscope, enabling all 'conventional' contrastmodes (phase, DIG, reflection, fluorescence) and selection of a sample area for examina-tion on nanometre scale by combined near-field optical and force microscopy. To thisend the sample stage has been modified to include a 100 x 100 um2 piezo-electric scan-ner. The adaptation stage at the right side contains optical and electronic components forprobe microscopy and supports the probe head. The probe head (shown tilted at ~ 45")can be rotated away from the sample to facilitate sample exchange and far-field inspec-tion. The probe head allows exchange of aperture-type fibre probes and cantilever-basedforce sensors for near-field optical, shear force, and normal force microscopy. Repro-duced by courtesy of Topometrix (35). a ••••• the base of the inverted microscope; b =bright-field illumination for conventional imaging; c = CCD camera; d = Ar+ laser sourceto be coupled in near-field fibre probe; e = sample stage with triangular-shaped large-area' piezo-electric scanner around objective; f = probe head, uplifted to facilitate accessto sample area; g = position of fibre probe and/or force sensor; h — adaptation stage,supporting probe head; i - microscope support with vibration damping.

Two protocols are given illustrating the more or less standardized steps infabricating the fibre probe (Protocol 1), and setting up the aperture-typeNSOM to examine a specimen (Protocol 2), It should be stressed that safetyprocedures to protect the eyes from damage should always be followed whenusing laser light.

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Protocol 1. Fabrication of an aperture-type near-field optical fibreprobe

Equipment• Optical fibre, preferably standard 125 um • Optical fibre/pipette puller, e.g. type P2000

diameter and single mode for the wave- of Sutter Instrum. Co. (37)length to be applied . Thin film aluminium coaler with film thick-

• Spectroscopic grade acetone and ethanol ness monitor (any type suffices, though the. Fibre cleaver (York Technology Ltd., e.g. sample holder should be modified to carry

type FK11) optical fibre which can be rotated)• High NA (> 0.7) optical microscope

Method

1. Cut a piece of fibre of sufficient length for tapering one end and cleavingthe other end (> 50 cm). Remove the plastic jacket at one end bymechanical stripping. Remove residual plastic using optical gradetissue rinsed in acetone. Wipe again using ethanol. A clean glass endis obtained by a final rinse in ethanol.

2. The next step, the tapering, is very critical. By local heating and con-trolled pulling the fibre will be tapered to end in a sharp apex. Thesequence of events during execution of a pulling cycle is as follows:

(a) The heat turns on.(b) The glass heats up and draws apart until a critical velocity, at

which point the heat is switched off.(c) After a delay a hard pull is performed to form the final tapered

fibre ends.

The taper angle close to the apex should be as large as possible toavoid optical loss in the final end with subwavelength dimensions.The Sutter P2000 fibre puller features CO2 laser heating (~ 10 W on0 1 mm) and allows reproducible setting of critical parameters such asheating power, heating profile, pull strength, velocity, and delay be-tween heating and pulling. Alternatively hot wire heating can beapplied, which requires a tightly coiled wire around the fibre toachieve sufficient heating for fibre melt.

3. Check the taper shape using a high resolution optical microscope. Anangle larger than 10° should be obtained while values approaching 30°are preferred. Iterate the pulling cycle to optimize the conditions.

4. The apex should be sharp, typically smaller than 50 nm, preferably witha flat end to facilitate the aperture formation. On optical inspection asharp tip will only show diffraction fringes. Any irregularity in thediffraction pattern surrounding the final end is an indication of roughstructure on a scale > 0.5 um. Regular diffraction fringes indicate a tipsize smaller than the diffraction limit, but give little information on the

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actual size. Only electron or ion microscopy will reveal the tip size andstructure. These elaborate inspection techniques are not performedroutinely, however, as they require tip coating and enhance the risk oftip damage.

5. In order to define an aperture and confine the light transmission to thedimension of this aperture the sides of the tapered fibre are thencoated with metal. Aluminium, with an optical penetration depth of 6nm, is most suitable for this purpose, where a thickness of 100-200 nmis sufficient for screening the light. The fibre is coated at 10-7 to 10-6 Twith high purity (> 99.99%) Al. The free end of the fibre should becoiled to fit in the coater. Evaporation coating at 70°-90° angle to thefibre axis is performed while rotating (~ 60 r.p.m.) the fibre. Thus theapex end is kept free and the aperture formed. The Al coating shouldbe free of grains. In this respect fast coating (10-20 nm/sec) of hot Al(preferably e-beam heated) from a source close to the fibre ( — 1 5 cm)is important. The film thickness is ideally measured using a quartzthickness monitor, while a shutter is important to set the coating time.

6. Finally the fabricated aperture fibre probe is checked by coupling lightinto the cleaved end of the fibre. Inspect the emitted light using a highresolution optical microscope to check for possible pin-holes in thecoating and irregular patterns in the emitted light. A good apertureprobe displays a single light spot surrounded by diffraction circles: theAiry pattern as determined by the inspection objective. A O ~ 100 nmaperture should transmit ~ 10 nW with a 10 mW laser coupled into thefibre. Additionally polarization characteristics can be examined. Elec-tron or ion microscopy will reveal the aperture size, but again, how-ever, there is the risk of losing the probe on inspection. Often theprobe characteristics are deduced from the performance in near-fieldoptical imaging.

The operation of an aperture-type NSOM with shear force feedbackinvolves:

(a) The approach of the fibre probe towards the sample surface until stableshear force feedback is established, very much as in conventional atomicforce microscopy based on cantilevered probes. Touching the samplesurface in NSOM, however, generally destroys the fibre probe, andreplacement with a new good aperture probe requires skilled experience.

(b) Optical alignment of the aperture with respect to the collection opticsand detector(s) is rather comparable to alignment procedures used inconfocal microscopy.

(c) Positioning the sample area of choice within the scan range of the piezo-electric scanners.

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Protocol 2. Fibre approach and optical alignment

Equipment• The presence of an aperture-type NSOM with shear force feedback is assumed

Method1. Mount a fibre probe in the scanning probe head of the microscope. Posi-

tion a sample or a bare substrate (for alignment) on the sample stage.Locate position and focus of the fibre and the sample area utilizing thefar-field imaging high NA objective. It is helpful in practice to illuminatethe fibre from the side, to produce a shadow that is easy to locate, and tocouple light into the fibre, which makes the aperture light up.

2. Using the coarse approach of the scanning probe head, manipulatethe fibre probe to the heart of the field of view, using lateral displace-ment, and to within 10 um from the sample surface.

3. Close the shear force sensing feedback loop with a high feedback gain,such that the vertical piezo element is at the limit of its range, corre-sponding to minimized tip-sample distance. Adjust the initial set-pointof the feedback loop at a value corresponding to weak shear forceinteraction, i.e. long working distance. Using manual or motorized fineadjustment, bring the tip close to the sample surface slowly enoughfor the feedback loop to respond promptly when the interaction comeswithin the range of the feedback interaction.

4. Optimize the shear force feedback loop by adjusting gain, set-point,mechanical fine adjustment, and optional frequency filters. This pro-cedure depends on the specific electronics, the scanning speed, thescan area, and on the topography of the sample. Generally some initialscanning while optimizing is required.

5. The light emitted by the aperture should be imaged confocally ontothe detector, which may be a pin-hole followed by a photomultipliertube or a small area avalanche photodiode. Optimize the final focusingand the lateral alignment of the aperture relative to detector. Filters orpolarization optics in the detection path allow fluorescence contrast,polarization contrast, etc.

6. Through the whole procedure it should be kept in mind that the probeis operated at a distance of some nanometres from the sample surface.Although best avoided altogether, manual adjustment of the system,while in shear force feedback, can be carried out, but with great caution.In mechanical shifting to a new sample area the probe should beretracted (out of feedback range) to a safe distance. The optical align-ment is not affected by shifting the sample.

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3. Applications3.1 Single fluorophores and proteinsAt the fundamental level of high resolution light microscopy is the observa-tion of individual molecules. The exact determination of the molecular loca-tion and its dipolar orientation is of interest because many dynamic biologicalprocesses, such as protein and molecular conformational changes, can bestudied by tracking the position and particular orientation of the molecules.Moreover, the single molecular response provides a sensitive tool to study thelocal environment of a single molecule at biologically relevant conditions. Forinstance, the exactness of fluorescence energy transfer experiments between adonor and acceptor pair depends critically on their relative distance andmolecular dipole orientation. In addition, if molecules are selectively excitedaccording to their particular orientation, it can be exploited as a tool to triggeror inhibit specific biological reactions. To date, light microscopy of singlemolecules at ambient conditions with high spatial and temporal resolution isreadily achievable by ultrasensitive fluorescence detection in a confocalarrangement (38). In 1993 Betzig and Chichester (26) were the first to imagesingle molecules using the near-field method. Molecules could be localizedwithin a few nanometres and their orientation in three-dimensions couldbe determined using polarization contrast. This achievement was directlyfollowed by single molecular spectroscopy (27) and single molecular fluor-escence lifetime detection (28).

Here a typical example of single molecular photodynamics as observed innear-field fluorescence by Garcia-Parajo et al. (39) is presented. A sampleconsisting of carbocyanine (DiI-C18) fluorophores embedded in a thin poly-methyl-methacrylate (PMMA) layer was prepared by spin coating a 5 X 10-8M concentration of DiI (Molecular Probes, D-282) molecules in methanol,added to a 0.5% weight PMMA in chloroform, onto a freshly cleaned cover-slip, resulting in a 5-10 nm layer with a surface coverage of typically a few dyemolecules per square micrometre. A time sequence of seven fluorescence a-NSOM images displaying single Di molecules dispersed in PMMA is pre-sented in Figure 5. An area of 1.5 X 1.5 um2 is scanned with 10 min interval. Amaximum fluorescence signal of ~ 5000 counts/sec is detected. Singlemolecules with a full width half-maximum (FWHM) of approx. 100 nm areeasily discriminated. The observed 100 nm spatial resolution is limited by theaperture of the probe used. In order to observe single molecular rotationalmovement the near-field scanning optical microscope is extended with twopolarization detection channels, allowing fluorescence to be detected simul-taneously at 0° and 90° polarization. In time, changes in the fluorescence ratiobetween both polarization channels are observed from frame to frame forthe molecules marked (a) and (b). The ratio signal is a direct measure forthe orientation of the molecular dipole moment. Additionally the signal

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Figure 5. Time sequence of near-field fluorescence images (1 to 7) displaying single car-bocyanine molecules (a and b) dispersed in PMMA. An area of 1.5 x 1.5 um2 (200 x 200pixels, 10 msec/pixel) is scanned with 10 min interval, while detecting single molecularfluorescence simultaneously at 0° and 90° polarization. Slow rotation of the molecules (a)and (b) can be observed in the changing fluorescence ratio between both polarizationchannels, until the discrete molecular photodissociation in image 5 and 7, respectively.Reproduced from ref. 39 with permission.

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Figure 6. Near-field fluorescence image (600 x 600 nm2) of single allophycocyan (ARC)trimers dispersed on a glass coverslip. The FWHM of the individual molecules is 100 nm,which is limited by the aperture of the probe used. The extrapolated signal for position-ing the probe above a peak is 5000-10000 counts/sec, which is sufficient to carry outspectroscopic measurements on a single protein basis. Reproduced from ref. 40 withpermission.

fluctuations are indicative of variations in the local molecular photophysics. Aunique sign of single molecular detection is the discrete molecular photodisso-ciation of molecules (a) and (b) in image 5 and 7, respectively (39).

Dunn et al. (40) have pioneered the near-field approach to image individualphotosynthetic proteins. Allophycocyan (APC) was chosen as one of thephycobiliproteins, contained in light harvesting protein complexes, that facili-tate the collection and funnelling of light energy towards reaction centres.APC has a trimeric structure, where each of the monomer units contains twoopen chain totrapyrrole chromophores, covalentty bound to the protein struc-ture, with strong absorption and emission bands in the visible regime. Asample of cross-linked APC trimers was prepared by spin coating a 5 X 10 8 Mconcentration in 0.1 M of phosphate buffer on a cleaned coverslip. Figure 6shows a 600 x 600 nm2 fluorescence a-NSOM image of single APC trimers.Distinct features with a width of approx, 100 m are clearly discernible, againlimited by the diameter of the tip. The actual individual trimers are 11 nm indiameter. Slight variations are observed in the emission intensities probablycaused by the random orientation of the chromophorc dipoles. No bleaching

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was observed, reflecting the photoslability of the APC trimer. The extrapo-lated signal for positioning the probe above a peak is 5000-10000 counts/sec,which is sufficient to carry out spectroscopic measurements on a single proteinbasis. This achievement demonstrates the feasibility of studying energy trans-fer dynamics on single molecule pairs (40).

3.2 Monolayers and aggregatesA Langmuir-Blodgett film is a highly organized and oriented mono-molecularfilm. Generally these films serve as model systems for molecular organizationin (bio)chemical membranes. For near-field optical microscopy, Langmuir-Blodgett films are practical test samples because of their homogeneous sur-face with nearly no topography and their well-defined molecular orientation.

Monolayers of diethylene glycol diamine pentacosa-diynoic amide (DPDA)(25), and 10, 12-pentacosa-diynoic-acid (PCA) (30), prepared by Langmuir-Blodgett technique, have been investigated. After UV polymerization thelayers were transferred to a glass substrate (41). Depending on the lateralpressure during polymerization and the transfer procedure, several uniformpolymerized domains were formed, with a wide range of lateral dimensions,but all with the molecular thickness of 6 nm. The monolaycrs display strongabsorption around \ = 500 nm and fluorescence at X = 550-600 nm, whereabsorption and emission dipole moment are along the highly oriented poly-carbon backbone.

A combined PSTM/AFM scan of a 1 X 1 um2 area of a PCA film is shownin Figure 7. The force image (Figure 7a) displays the monolayer topography of

Figure 7 PSTM/AFM image (scan area 1 x 1 um2:) of a 10, 12-pentacosa-diynoic-acid (PCA)Langmuir-Blodgett monolayer on a glass substrate. Reproduced from ref. 30 with permis-sion, (a) Force image displaying the topography of monolayer patches, 6 nm thick anddimensions of a few 100 nm. (b) Simultaneously obtained PSTM image displaying absorp-tion of the excitation at \ = 514 nm. Edge steepness of the optical contrast is 30 nm.

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6 nm height, as contained in the z-piezo signal in force feedback mode. Thecorresponding photon tunnelling image (Figure 7b) displays the fraction ofthe incident p-polarized light of the Ar+ line at 514 nm which is directed tothe detector by the local coupling of a dielectric SiN probe (Figure 2, bottom).Monolayer domains with an area smaller than a square wavelength are clearlyvisible in the photon tunnelling image. Clearly the lateral resolution is beyondthe diffraction limit, with an edge steepness of 30 nm. The PSTM signal on thedomains is 10% below the signal detected on the surrounding glass, which isin agreement with the measured absorption of a PCA monolayer for greenlight by far-field methods. Consequently, the PSTM contrast is mainly causedby absorption for this sample (30). The influence of the topography on theoptical coupling is limited for this specific thin sample, however for thickerbiological samples the effect of topography and far-field scattering becomesdominant (29).

An a-NSOM image of a 4 X 4 (um2 area of a DPDA film is presented inFigure 8. In the shear force image (Figure 8a) several domains and the under-lying glass substrate are visible. Figures 8b and 8c show the correspondingnear-field fluorescence images, where the incident linear polarization isdirected perpendicular between the two images. Peak value of the fluores-cence intensity is about 105 photons/sec. The absorption and emission dipolemoments of the polydiacetylenes are oriented parallel to the polymer back-bone, where the orientation is uniform over each domain due to the crys-tallinity of the Langmuir-Blodgett film. The near-field fluorescence imagesclearly demonstrate the high anisotropy of the polymerized diacetylenic filmswith ~ 100 nm lateral resolution: domains which are fluorescent for a givenpolarization direction in one image are dark for the perpendicular polariza-tion direction in the other image. The force image displays the topography ofthe monolayer domains with a lateral resolution of ~ 30 nm, showing somesurface roughness and a few non-fluorescent structures. Comparison of theseimages clearly demonstrates the advantage of near-field optics in combinationwith force microscopy: the near-field optical images allow determination ofthe polycarbon backbone orientation and polymerization efficiency, addi-tional to the topography in the simultaneously recorded force image, bothwith high lateral resolution (25).

Thin films of J-aggregates are also interesting model systems for study ofmolecular orientation and ordering at nanometre scale. The strong couplingof the dipoles in J-aggregates over many nanometres causes large spectralshifts and narrowing of bands with peculiar exciton dynamics, which makesthem specifically interesting subjects for near-field optical microscopy. Thegroup of Barbara (42) has performed extensive studies of J-aggregates bynear-field fluorescence microscopy. Interest was focused on J-aggregates ofpseudoisocyanine (PIC) grown in thin poly(vinyl sulfate) (PVS) films onquartz substrates, which display a fibrous structure along several hundredsof microns with about 150 nm width and 10 nm thickness. Absorption and

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Figure 8. A 4 x 4 um2 scan of a Langmuir-Blodgett monolayer: UV polymerized diethyleneglycol diamine pentacosa-diynoic arnide, DPDA. Reproduced from ref. 25 with permission,(a) Shear force image, showing the topography of 8 nm monolayer patches on the glasssubstrate. (b) and (c) Near-field fluorescence images with mutually perpendicular direc-tions of linearly polarized excitation, showing the high anisotropy of the Langmuir-Blodgettfilm and indicating the orientation of the polymer backbone.


b)

Position um)

Figure 9. Near-field fluorescence images (5 x 5 um2) of PIC J-aggregates in thin PVSfilms excited with 570 nm light from a a-NSOM probe. Reproduced from ref. 42 with per-mission, (a) and (b) Excitation light from the probe polarized vertical and horizontal (onthe page) respectively, with all polarization directions of fluorescence detected. Thestrong polarization dependence of direct excitation to the lowest energy excitonic state ofthe aggregate is clearly observed, (c) Line scans across several of the aggregate fibres of(a) and (b).

emission of these PIC aggregates peaks sharply at 573 nm, the so-calledJ-band corresponding to transitions to the lower edge of the excitonic hand.Fluorescence a-NSOM images for linearly polarized excitation at \ = 570 nmand detection at \ > 590 nm are presented in Figure 9. Both polarization per-pendicular and parallel to the scan direction are shown in Figure 9a and 9b.respectively, while all polarization directions of fluorescence arc detected. Astrong polarization dependence of direct excitation to the lowest energy exci-tonic state of the aggregate is clearly observed in the preferentially verticallyand horizontally aligned fluorescent fibres in Figures 9a and 9b, respectively.

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The line scans across several of the aggregate fibres in Figure 9c shows extinc-tion ratios up to 9:1. The local transition dipole is along the long axes of theaggregates, both for absorption and emission. In contrast to this, fluorescenceexcitation images recorded at lower wavelength (488 and 514 nm), i.e. higherenergy excitonic transitions, show little dependence on the excitation polariz-ation with rather a slight preference for polarization perpendicular to the longaxis of the aggregates. From these combined spectroscopic and polarizationdata a herringbone ordering of monomers within the aggregate could be con-cluded. Moreover, the high spatial resolution sets an upper limit of ~ 50 nm tothe scale of excitonic energy migration (42). Clearly the unique combinationof spectroscopic, polarization, and topographic data, all on nanometre scale,reveals details inaccessible to far-field light microscopy.

3.3 VirusViruses are generally not accessible by conventional optical microscopy andin consequence an interesting object of study for the near-field alternative, todemonstrate the high resolution potential in biological applications. A well-known candidate is the tobacco mosaic virus (TMV), a rod-shaped virus about300 nm long and 18 nm in diameter, with a central hole of 4 nm diameterenclosing a single-stranded RNA molecule. The TMV protein subunits packto form a helix with a well-known structure. Indeed TMV was one of the firstobjects used to test the a-NSOM of Topometrix Corp. (43). TMV particleswere attached to amino-silanated atomically flat mica by glutaraldehyde, andimaged after rinsing and drying. Individual TMV particles could be resolvedby this technique with separations of less than 30 nm. Interestingly the TMVparticles displayed higher optical transmission than the supporting mica sub-strate, probably caused by the difference in refractive index, but more likelyinduced by the distance regulation on the TMV topography. Detailed near-field optical imaging of isolated viruses is an area yet to be explored.

3.4 Cellular surface and cytoskeletonCellular structures have been the object of study for conventional opticalmicroscopy ever since the earliest days of microscopy. For scanning probemicroscopy cells are rather large objects, where generally only the surface canbe studied. AFM studies have yielded however a wealth of information on cellmembrane structure (pores (10), antigens (44), and proteins), mechanical cel-lular properties (membrane hardness, visco-elasticity) (9), and the temporalbehaviour of living cells (interaction, growth, formation of pseudopodia) (9).Occasionally, subsurface structure is obtained by applying high forces (> 10 nN)in contact force microscopy (10) such that the cytoskeleton shows up whilethe membrane is locally pressed down. The spectrum of structural informa-tion can be extended using specific labelling techniques (immunogold (44),enzymes (45), or fluorescence), as they have been developed for electron

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microscopy and (confocal) fluorescence microscopy. Clearly fluorescent labelsoffer interesting potential for near-field fluorescence detection with its singlemolecular sensitivity. As such fluorescence NSOM forms a bridge techniquebetween AFM and confocal fluorescence microscopy, because in addition tothe surface topography specific cell surface and skeletal constituents can belocalized with subdiffraction resolution through their fluorescence.

In 1993 Betzig et al. (24) showed the first application of near-field fluor-escence microscopy to labelled cytoskeletal actin in fixed mouse fibroblastcells. Hereto Swiss mouse 3T3 fibroblast cells were fixed in formaldehyde andair dried on a glass coverslip, after specific fluorescent staining of filamentousactin with rhodamine-phalloidin. Figure 10 shows images of a cellular pro-trusion: a flat thin lamellipodium of the 3T3 cell. Conventional and scanningconfocal fluorescence images, both with X 100, NA 1.3 objective, are pre-sented in Figures 10A and 10D, clearly showing the effect of reduced detectionvolume in confocal detection. Figures 10B and 10E are 10 X 10 um2 magnifi-cations of the lamellopodium area to facilitate comparison with the shearforce (Figure 10C) and near-field fluorescence images (Figure 10F) of thesame region. Clearly, the force image is distinctly different from the near-fieldfluorescence image although they were acquired simultaneously. The forceimage shows mainly surface topography with about 50 nm lateral resolution,whereas the near-field fluorescence image shows the cytoskeleton organiza-tion in fine detail with a lateral resolution of about 100 nm, well beyond thediffraction limited resolution of the confocal fluorescence image. The near-field fluorescence contrast is similar to that seen in confocal fluorescenceimages, easy to interpret, though with improved resolution, and almostbleach-free. Yet, it should be noted that the near-field fluorescence mainlyoriginates from the cellular volume within about the first 100 nm below thecell surface, while the confocal section as generally obtained is fluorescence ofthe plane of focus.

3.5 Chromosomes and fluorescence in situ hybridizationDrosophila polytene interphase chromosomes are a classical object of studywith phase-contrast optical microscopy, because of their relatively large sizecompared to human chromosomes and their characteristic natural band pat-tern of alternating dark bands and lighter interbands. Both bands and inter-bands contain fibre-like structures, where bands are composed of denselycoiled fibres, while interbands display more dispersed fibrils with a highercontent of DNA and protein. The fibre width is typically 10-30 nm, which isassociated to the first winding of the DNA double helix around histones form-ing a 11 nm thick nucleosome fibre and the subsequent coiling of the nucleo-somes into a 30 nm thick solenoid fibre. Polytene chromosome substructurehas been extensively studied by electron microscopy and more recently alsoby AFM (46, 47). The band structure is revealed as strong topography in

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Figure 10. Six images of a stained single lamellipodium from a Swiss mouse 3T3 fibro-blast cell. Reproduced from ref. 24 with permission. (A) Conventional fluorescence imagewith a x 100, 1.3 NA objective. (B) Magnified image of the boxed area in (A). (C) Shearforce topographic image of the same area. (D) Confocal fluorescence image with thesame x 100, 1.3 NA objective. (E) Magnified confocal image from (D). (F) Near-field fluor-escence image. The NSOM image combines resolution superior to confocal microscopywith contrast that provides more detail than in force microscopy.

AFM data, as is clearly illustrated in Figure 11. Figure 11a displays a largearea AFM scan of a Drosophila polytene chromosome after fixing, squashing,and air drying on an object glass, as it was imaged at TopoMetrix Corp. (35).The correspondence with the well-known optical picture is striking, where inthe AFM image the bands are high and interbands lower. Figure 11b shows amore detailed scan of an interband region, revealing 10-30 nm fibres with avariable extent of coiling, especially when extending into the band area.Finally Figure 11 shows the results obtained with the TopoMetrix Auroraa-NSOM obtained on the same chromosome sample, with the correspondingshear force and near-field 'bright-light transmission' image in Figures 11c andlid, respectively (35). The high correlation between the force and near-fieldimage are an indication for topographic-induced contrast in the near-fieldoptical images. Yet, also differences are observed, e.g. the stretched fibresappear bright in transmission, while the condensed coils appear dark. Again abetter defined high resolution contrast is expected by monitoring near-fieldfluorescence.

Obviously, human chromosome structure has been investigated with all themicroscopic techniques available. In addition to the pure morphological ex-amination of chromosomes, specific information on the DNA sequence canbe correlated to the chromosome structure by the use of labelling techniques,specifically in situ hybridization as developed almost 30 years ago (48). Espe-cially optical detection of in situ hybridized DNA, on the basis of fluorescencelabels or enzyme generated dyes, has promoted fluorescence in situ hybridiza-tion (FISH) to one of the major cytogenetic detection methodologies forhuman genetics (49). FISH enables direct visualization of topological or posi-tional information of gene sequences in a fluorescence microscope, allowingrapid localization of genomic DNA fragments in morphologically preservedinter- and metaphase chromosomes. A resolution better than 106 base pairscan be obtained using (pro)metaphase chromosomes (50), while 1 kb is feasibleon stretched DNA. The resolution of the fluorescence labels is fundamentallylimited to ~ 300 nm by diffraction in conventional fluorescence microscopy,yet localization of the numerous closely linked genes requires mapping at ahigher resolution. Using electron microscopy immunogold DNA probes canbe imaged with nanometre resolution. Putman et al. (45) pioneered the poten-tial of atomic force microscopy (AFM) in the detection of morphological insitu hybridization labels, and were able to discriminate morphological labels

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Niek F, van Hulsl

Figure 11. Four images of a Drosophila polytene chromosome after fixing, squashing, andair drying. Reproduced by courtesy of Topometrix (35). (a) Large area (150 x 150 um2)AFM scan revealing the band structure as high bands and low interbands, with remark-able correspondence to the well-known optical picture, (b) A more detailed AFM scan(12 x 12 um3) of an interbank region, revealing 10-30 nm fibres with a variable extend ofcoiling, (c) Shear force topographic surface plot (7.5 x 7.5 um2). (d) Corresponding near-field 'bright-light transmission' image.

of 75-100 nm diameter, after enhancement by an enzymatic reaction, howeverwithout observing single copy DNA targets. Despite the higher resolution ofelectron and force microscopy they lack the multiplicity of fluorescence detec-tion. In fact, the widespread use of FISH is mainly due to its unparalleledspecificity afforded by the potential of multicolour labelling in one prepara-tion (50). Near-field fluorescence microscopy has the potential to combine thebest of both: optical resolution beyond the diffraction limit and multiplicitythrough multicolour fluorescence detection with sensitivity down to thesingle molecular level. Moers et al. (51) have explored near-field two-colourfluorescence detection of FISH labels on human metaphase chromosomes,combined with the chromosomal morphology as obtained by shear forcedetection. Some results are presented below.

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Figure 12 shows a scan of a human metaphase chromosome 1, with specificlabelling of the telomeric region (top of the image). The metaphase chromo-some spread was prepared on a microscope coverslipv according to routineprocedures. The repetitive DNA probe pl-79 was used, which is specific forthe telomere region (1p36) of the short arm of chromosome 1, with an insertsize of 0.9 kb. The probe pl-79 was labelled with digoxigenin and detected bycyanine (Cy3, an orange fluorescent dye) (51). The pixel size is 35 nm. Thescan speed is 40 msec/pixel. In the shear force image (Figure 12a) the highspatial frequency filtered piezo feedback signal is displayed, revealing thewell-known metaphase chromosomal structure with well-separated chromatids,details as small as 40 nm and height up to ~ 150 nm. The corresponding near-field fluorescence image (Figure 12b) displays the green fluorescence at X >570 nm, using BG39 and KV550 Schott filters, with excitation by the 521 nmKr+ line. The pl-79 probes are visualized as distinct substructure in thetelomere region with at least five probes in each chromatid (maximum700 counts/pixel). Some bright fluorescent material, not associated with thechromosome, is detected on the glass substrate in the right side of Figure 12b.In addition to the bright signals at the telomere several isolated weak spots(—50 counts/pixel) are detected on the chromatids in the centromeric region,as presented in a magnified view in Figure 12c. These labels are most probablydue to non-specific hybridization of pl-79. Figure 12d shows a 1 um line tracethrough two weak Cy3 labels as indicated in Figure 12c. Both fluorescencespots have a width of 80 nm, while separated at a distance of only 110 nm. Theline trace clearly indicates the superior optical resolution provided by NSOM,including the ability to localize the fluorescence maximum with an extremeaccuracy of a few nanometres. The signal level of the fluorophores in Figure 12dcorresponds to ~ 1000 counts/sec, which is estimated to originate from lessthan ten Cy3 molecules. Evidently the single molecular sensitivity is retainedalso when imaging 'large scale' biological structures, such as chromosomes.

4. ConclusionsNear-field scanning optical microscopy is a true optical microscopic techniqueallowing fluorescence, absorption, and polarization contrast with the addi-tional advantage of nanometre lateral resolution, unlimited by diffraction.Especially fluorescence NSOM gives a clear high resolution contrast andinduces virtually no bleaching as opposed to confocal fluorescence micro-scopy. Bright-field NSOM in transmission generally yields a complicated con-trast caused by a mixture of dielectric and topographic contributions. Shearforce feedback is essential for reliable operation of aperture NSOM based onfibres, especially while scanning over soft surfaces of cells and chromosomes.The force feedback gives a topographic map of the sample surface simultane-ously with the near-field optical image. Also photon tunnelling microscopy in

367

Figure 12. Human metaphase chromosome 1, with specific labelling of the telomericregion (top). Scan area 7 x 7 um2, with 35 nm pixel size and 40 msec/pixel. Reproducedfrom ref. 51 with permission, (at Shear force image, high pass filtered in horizontal direc-tion, showing chromosomal structure with details as small as 40 nm and height up to- 150 nm. (b) Corresponding near-field fluorescence image, displaying green Cy3 fluor-escence with at least five probes in the telomere region of each chromatid. (c) Magnifiedview of the centromeric region of the near-field fluorescence image, showing several iso-lated weak spots. (d) A 1 (um line trace through two weak Cy3 labels as indicated in (c). Bothfluorescence spots have a width (FWHM) of 80 nm, while separated at a distance of only110 nm, which clearly illustrates the superior lateral optical resolution provided by NSOM.

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combination with force microscopy allows routine scanning with a high opticallateral resolution, however interference effects can be dominant on surfaceswhich display extensive scattering. As such, in contrast to a-NSOM, theapplication potential of PSTM to biological surfaces is rather limited.

Several biological applications of near-field optical microscopy, in combina-tion with force microscopy have been presented. It is shown that apertureNSOM with fluorescence detection gives (bio)chemical specificity and orien-tational information, in addition to the simultaneously acquired topographicalimage. On the microbiological scale the technique has large potential forDNA sequencing and molecular organization on membranes. At the cellularlevel it allows study of the role of the cytoskeleton in cellular mobility in cellgrowth, cell migration, formation of protrusions, etc.

5. Future outlookNear-field optics is gradually becoming mature and moving from the develop-mental stage towards applications. The first commercial instruments haveappeared on the market, yet the operation requires a rather specialized andexperienced microscopist.

The aperture probe brightness of ~ 10 nW is sufficient for single moleculardetection, yet is still rather limited for spectroscopic applications, such as fluor-escence and Raman, where photon noise will be a fundamental limit in the speedof imaging. The lateral resolution is 20-100 nm, almost an order of magnitudebeyond the confocal microscopic resolution, yet still rather poor compared toresolution achieved with force microscopy. Ongoing research on probe fabrica-tion through optimized fibre pulling and etching or micro-machining may resultin future probes with higher efficiency, versatility, and resolution.

All biological applications presented have been obtained on dry samples.Imaging at physiological conditions obviously requires operation in liquid. Inprinciple near-field optical probes can be made to function in liquid, just likeforce sensors, however reliable imaging on soft material is rather delicate andyet to be developed for the optical alternative.

Finally the monitoring of biological processes requires the development ofhigh frequency scanning and high efficiency probes for sufficiently fast imageacquisition.

For further information on recent advances in the development and appli-cation of near-field optical microscopy the reader is referred to the Proceed-ings of the international conferences on near-field optics (52, 53).

AcknowledgementsImages were generously made available by: Paul West, Gary Williams, andWouter Rensen of TopoMetrix Corp., Santa Clara, California; Paul Barbara,

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NiekF. van Hulst

Daniel Higgins, and Chuck Tomlinson of University of Minnesota, Minne-apolis; Sunney Xie of Pacific Northwest Labs, Richland, Washington; EricBetzig of NSOM enterprises.

The author thanks Ton Ruiter, Marco Moers, Joost-Anne Veerman, MariaGarcia-Parajo, Wouter Rensen, Kees van der Werf, Frans Segerink, EricSchipper, Ine Segers, and Bart de Grooth, all of the Applied Optics group atUniversity of Twente, The Netherlands, for numerous data, their assistance,and suggestions.

This research is supported by the European Human Capital & Mobility net-work on Near-field Optics and Nanotechnology and the Dutch Foundationfor Fundamental Research (FOM).

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Introduction of materials intoliving cells

1. Particle bombardment as a means of DNA transferinto plant cellsCHRISTIAN SCHOPKE and CLAUDE M. FAUQUET

1.1 IntroductionSince its inception a decade ago, particle bombardment, also known as the'Biolistic process' (1), has developed into a method of considerable import-ance in many areas of biology (see Table 1). It has been especially successfulin the genetic transformation of plants, where the presence of a cell wall posesa significant obstacle for the introduction of large molecules, including DNA.The purpose of the first part of this chapter is to illustrate the use of particlebombardment for the introduction of DNA into plant cells and the utilizationof reporter genes to follow the fate of genetically transformed cells, using cas-sava (Manihot esculenta) as an example. The reader who is interested in moredetailed information on particle gun types and on different applications ofparticle bombardment is referred to several recently published reviews (2-4).

Table 1. Examples for the application of particle bombardment

aIn this case bacterial magnetic particles were used for bombardment.

10

Target

MitochondriaChloroplastsCyanobacteria"FungiPlantsPlantsMammalian cellsand tissues, liveanimals

Species

Saccharomyces cerevisiaeTobaccoSynechococcusPaxillus involutesMore than 20 important cropsCucurbitaceaeVarious

Result Reference

Stable transformation 5Stable transformation 6Stable transformation 7Stable transformation 8Stable transformation 9Virus infection 10Transient expression, stable 11transformation, immunization

Christian Schopke, Claude M. Fauquet, and H. F. Paterson

1.2 Practical considerationsThe following discussion will focus on a particle gun type that has been usedroutinely by many laboratories, the particle delivery system PDS-1000/He(Bio-Rad) (see Figures 1 and 2). In this device pressurized helium is used toaccelerate a macrocarrier onto which DNA coated microparticles have beenapplied. The accelerated macrocarrier then hits a stopping screen, permittingonly the particles to continue their flight. The momentum imparted to theparticles is such that they can penetrate plant cell walls (12). In a small pro-portion of cells that receive the particles (in the range of 100-2000 cells/cm2 ofbombarded surface area), transient expression of the introduced gene can beobserved. The number of cells that eventually incorporate the introducedDNA into chromosomal DNA is in the range of 0.1-1% of transientlyexpressing cells,

1.2.1 Parameters that affect the success of particle bombardmentNumerous factors can have an influence on the outcome of a bombardmentexperiment (4, 13). If a new type of tissue is to be used for particle bombard-ment, a good strategy is to start with the basic settings given by the manufac-turer of the PDS-1000/He (similar to Protocol 3, step 4) and to optimize

Figure 1. The particle delivery system PDS-1000/He.

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10: Introduction of materials into living cells

Before After

Figure 2. Schematic representation of the PDS-1000/He system before and after activa-tion (not to scale). A, B, and C are the adjustable distances that influence the velocity withwhich the microcarriers hit the target cells. (A) Rupture disk-macrocarrier distance.(B) Macrocarrier travel distance . (C) Target distance. The arrows indicate the direction ofthe helium flow (from ref. 12, modified with permission from Bio-Rad).

parameters, including bombardment pressure, distance between the barreland tissue sample, number of bombardments per sample, and particle size. Itis very important that the tissue is in a favourable physiological state to with-stand the stress related to bombardment. In many cases culture of the tissuebefore and after bombardment on a medium containing elevated concentra-tions of osmotically active substances results in higher rates of transient geneexpression. For a thorough discussion of factors that influence the efficiencyof particle bombardment the reader is referred to refs 14 and 15.

1.2.2 Particle coatingThe basic protocol for coating microparticles with DNA consists of mixing asterile suspension of tungsten or gold particles in water (see Protocol 1) withDNA, CaCl2, and spermidine. Numerous variations of this procedure havebeen employed, e.g. with differences in total amounts of DNA and particlesused, the ratio of DNA to particles, the addition of buffers to the particle sus-pension, and the extent of the use of sonication and/or vortexing to mix thesuspension. In the case of the PDS-1000/He, in the final step of the coatingprocedure the particles are resuspended in 100% ethanol. Aliquots of thissuspension then are transferred to macrocarriers. After drying they are readyto be used for bombardment. At the International Laboratory for TropicalAgricultural Biotechnology (ILTAB) we routinely use with good results aprotocol based on that developed by Sivamani et al. (16) (see Protocol 2).

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Protocol 1. Sterilization of particles

Equipment and reagents• Eppendorf microcentrifuge 5415C (Fisher • Gold particles (Bio-Rad or Alfa Chemicals)

Scientific Co.) or tungsten particlesb (Bio-Rad)• Microcentrifuge tubes, 1.7 ml, polypropy-

lene, siliconized" (Sigma, T-3406)

Method

1. Weigh 30 mg particles into a microcentrifuge tube.

2. Add 500 u| 100% ethanol and vortex for 3 min at maximum speed.

3. Centrifuge for 10 sec at 10 000 r.p.m.

4. Remove supernatant and add 500 ul 100% ethanol.

5. Vortex for 2 min at maximum speed.

6. Repeat steps 3-5.

7. Centrifuge for 10 sec at 10000 r.p.m.

8. Discard supernatant and add 500 ul sterile distilled water.

9. Vortex for 2 min at maximum speed.

10. Centrifuge for 30 sec at 10 000 r.p.m.

11. Discard supernatant and add 500 u| sterile distilled water. The particlesare now ready for coating.c

aThe use of siliconized tubes is recommended because the particles tend to stick to the surfaceof non-siliconized tubes during the following manipulations.bIn many publications on particle bombardment the source for tungsten particles is given asGTE (now Osram Sylvania, Danvers, Massachusetts).c Gold particles should be prepared the day they are to be used because they agglomerate irre-versibly over time in aqueous suspension (4). Tungsten particles should not be stored forlonger than one to two weeks as the oxidation of the surface of the particles may negativelyaffect the DNA binding capacity (4).

Protocol 2. Coating of particles with DNA

Equipment and reagents• See Protocol 1 • 0.1 M spermidine, free base (Sigma, S 4139)• Macrocarriers (Bio-Rad) • DNA at a concentration of 1 ug/ul in water. 2.5 M CaCI2

Method

1. Resuspend a sterile suspension of gold or tungsten particles (seeProtocol 1, step 11) and transfer an aliquot of 50 uJ containing 3 mgparticles to a siliconized microcentrifuge tube. While vortexing the

376


open tube on a low setting, add 5 ul DNA solution (1 ug/uj). Vortexcontinuously through steps 2-3.

2. Add 20 ul 0.1 M spermidine.3. Add 50 ul 2.5 M CaCI2, drop by drop.4. Let the particles settle for 10 min at room temperature.

5. In the meantime sterilize the macrocarriers and the macrocarrier holdersby leaving them for 10 min in 100% ethanol. Remove macrocarriersand macrocarrier holders from the ethanol, let them dry, and insert themacrocarrier in the holders. Store them in sterile Petri dishes until theyare used. We routinely prepare ten holders at a time for use up to 1 hafter preparation.

6. Remove the supernatant from the settled suspension (step 4) and add50 (uJ cold 100% ethanol.

7. Vortex at a low speed to resuspend the pellet.

8. Distribute samples (usually in the range of 5-10 ul) onto the centre ofthe macrocarriers and let them dry in a desiccator. Leave them in adesiccator until they are used. While pipetting the suspension, it isimportant to maintain the pipette in a vertical position to ensure thatthe suspension spreads evenly on the surface of the macrocarrier.

1.2.3 The choice of a target tissueIf the purpose of particle bombardment is transient expression of the intro-duced gene, for example to evaluate the expression of transcriptional pro-moters (17), any tissue that remains viable over the course of the experimentcan be used as a target. However, if the production of stably transformed tissueis required, transformed cells must be able to undergo continuous cell division.While many plant cell types can resume mitotic activity and produce unorgan-ized callus under appropriate conditions, the capacity to regenerate into wholeplants usually is restricted to only a few cell types. The ideal target for the pro-duction of transgenic plants would be a leaf with an epidermis that contains ahigh percentage of single cells capable of regeneration. The flat shape wouldensure that the surface of the tissue is at right angles to the beam of acceler-ated particles, and the target cells would be in the cell layer that is hit byparticles with the highest efficiency.

In the case of cassava, regeneration can be achieved via organized embryo-genie structures (embryo clumps) derived from young leaves or via friableembryogenic tissue (maintained either as callus or as suspension culture)derived from somatic embryos. Both tissue types have been used as targets forparticle bombardment for the purpose of obtaining transgenic cassava plants(18-20) (see Protocols 3-5). However, they differ in important aspects withregard to particle bombardment and to regeneration patterns. Embryo clumpsare composed of tightly packed embryos at the globular to torpedo stage, i.e.

377


they have an epidermis and, depending on the developmental stage, a visibleshoot pole. They can be induced either to grow into plants or, alternatively, toform secondary embryos (21). Secondary embryogenesis proceeds through acleavage-like process in which many cells in layers below the epidermis areinvolved (22). On the other hand, tissue derived from embryogenic suspen-sions is composed mainly of small globular structures, 100-1000 u.m in diameter,which grow freely suspended in liquid medium. Secondary embryogenesis isinduced in single cells on the surface of the globular structures (23). When thistype of tissue is spread on a flat support such as filter paper, it comes close tothe ideal target mentioned above: a layer of tissue with a high percentage ofthe cells on the surface capable of regeneration.

Protocol 3. Particle bombardment of embryo clumps of cassava

Equipment• Biolistic particle delivery system PDS-1000/ • Micropipetters

He (Bio-Rad) • Macrocarriers (Bio-Rad)

Method

1. Establish cultures of embryo clumps of cassava as described in ref. 21.

2. Three to four weeks after their last subculture, these clumps consist ofmore or less globular embryos in their centre, torpedo-shapedembryos at their margins, and often some non-embryogenic callus.Remove this callus and arrange pieces of embryo clumps consistingmainly of globular embryos in a circle of 2 cm diameter (about 30clumps with a diameter of 3-6 mm) in the centre of a Petri dish withagar medium. It is important to choose clumps in which the embryosare arranged more or less horizontally in order to be accessible for theparticles during bombardment.

3. Prepare DNA coated particles (gold, 1 um diameter) according toProtocol 2.

4. Choose the following bombardment parameters:(a) Distance macrocarrier/stopping screen: 6 mm (upper position of screen).(b) Distance rupture disc assembly/macrocarrier cover (gap): 1/4"

(63.5 mm).(c) Suspension volume per bombardment: 7.5 ul.(d) Rupture disks: 650 psi.(e) Level of sample holder: third from below.

5. Place the Petri dish with the embryo clumps in the centre of the sampleholder. Bombard the sample under a partial vacuum of 10-6.5 kPa(vacuum of 27-28 in Hg).

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Protocol 4. Preparation of tissue derived from embryogenicsuspensions of cassava for particle bombardment

Equipment• Polypropylene grids, autoclavable, opening • 15 ml graduated polystyrene centrifuge

210 um (Spectrum Medical Industries, Inc.): tubes (Falcon, 2099)the grids can either be purchased as circles(5 cm diameter, order no. 145 771) or as 30cm2 sheets (order no. 146 428)

Method

1. Establish an embryogenic suspension of cassava as described in ref. 23.Take a sample of 10 ml with a wide-mouth glass pipette or with a plasticpipette that has been cut with a hot scalpel blade to obtain a wideopening and transfer it to a 15 ml graduated centrifuge tube.

2. Leave the suspension undisturbed for 20 min and read the settled cellvolume (SCV) of the tissue. Take an aliquot of 1 ml tissue and initiate anew suspension. Subculture every second day in fresh medium.

3. After 12-14 days, sieve the suspension to obtain the fraction ofembryogenic cell clusters with a diameter between 250-500 um.

4. Place the grids in Petri dishes on top of a dry filter paper. Transferaliquots of 200 ul SCV in a volume of 1 ml culture medium with awide-mouth pipette onto the grids in such a way that the liquid formsa drop kept in place by surface tension.

5. Prepare the amount of dishes needed for one bombardment session.Including the time for particle coating and tissue preparation, approx.40 samples can be bombarded in a period of 8 h. If each sample isbombarded twice, the amount of dishes is reduced correspondingly.

Protocol 5. Particle bombardment of tissue derived fromembryogenic suspensions of cassavaa

Equipment and reagents• Biolistic particle delivery system PDS- • DNA coated particles (see Protocol 7)

1000/Helium (Bio-Rad) . P|ant tissue (see Protocol 2• 1100 psi pressure disks (Bio-Rad)

Method

1. Use the bombardment parameters as described in Protocol 3, step 4.Use 1100 psi rupture disks and a volume of 5 uJ suspension of coatedparticles per bombardment.

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2. Immediately before bombardment, bring the droplet with tissue on thegrid in contact with the underlying filter paper. This can be done eitherby pressing down the grid with a forceps or by adding a drop ofmedium between the grid and the filter paper. As a result, the liquid isabsorbed and the tissue remains as an evenly spread layer on the grid.If the grid has changed its position during the manipulations, move itback to the centre of the dish.

3. Place the Petri dish with the tissue on the sample holder inserted at thethird level from below in the particle gun. Bombard the sample undera partial vacuum of 10-6.5 kPa absolute pressure (vacuum of 27-28in Hg).

4. After the bombardment, transfer the grid with tissue to a Petri dish orany other suitable vessel with liquid culture medium. At the end of thebombardment session transfer the tissue to a culture flask.

5. If the sample is to be bombarded a second time, add enough mediumto the dish so that the tissue is just covered. This ensures that it doesnot dry out during the preparations for the next bombardment.

6. For a second bombardment, transfer the grid to a Petri dish with afilter paper to absorb excess medium. Continue with steps 3 and 4.

a Adapted from ref. 19.

1.3 Identification of cells transformed with reporter genesSeveral genes have been routinely employed in the context of particle bom-bardment with the purpose of visually identifying transgenic plant cells ortissues. The expression of these genes can be detected either directly throughtheir coloured product or indirectly through a colour reaction catalysed by thegene product (see Table 2).

Table 2. Examples of reporter genes used in conjunction with particle bombardment

380

Gene(s) Origin Gene product Result of gene Referenceexpression

uidA.lucRand C1

gfp

E. coliFireflyMaize

Jellyfish

B-glucuronidase (GUS)Luciferase (LUC)Enzymes involved inanthocyanin synthesisGreen fluorescent protein(GFP)

Blue stainLight emissionExpressing cells red

Fluorescence

242025

26


1.3.1 The B-glucuronidase gene (uidA)uidA is a gene isolated from Escherichia coll and encodes the enzyme (3-glucuronidase (GUS), which can be detected by a colour reaction (Protocol6). This enzyme catalyses the cleavage of B-glucuronides into glucuronicacid and the corresponding aglycone. The substrate used most frequently forhistological purposes is 5-bromo-4-chloro-3-indolyl-p-D-glucuronide cyclo-hexyl-ammonium salt (X-gluc). The reaction with B-glucuronidase results inglucuronic acid and the colourless, water soluble 5-bromo-4-chloroindoxyl,which upon oxidative dimerization forms an insoluble blue stain. The local-ization of the blue stain depends on the diffusion of the water solublemonomer, 5-bromo-4-chloroindoxyl, and on the velocity of the dimerizationreaction. The longer this reaction takes, the farther away from its place oforigin the monomer can diffuse. This means that, depending on the assayconditions, the blue stain can be formed in cells adjacent to the cell thatreceived the uidA gene and thus reduce the resolution of the assay. Thehistological GUS assay has been widely used for experiments to optimizemicrobombardment parameters. In the context of particle bombardment, theGUS assay can be used to determine the number of cells transiently expres-sing the uidA gene, and to optimize each experimental system. When embryo-genie tissue of cassava was bombarded with uidA and analysed by standardGUS assay conditions (24), it was found that two to three days after bom-bardment large areas of tissue were stained light blue (27). With increasingconcentrations in the assay buffer of equimolar mixtures of the oxidationcatalysts, ferri- and ferrocyanide, the blue stain became more intense andrestricted to smaller areas, and at a concentration of 6.4 mM each most of thestain was confined to single cells (Figure 3A, 3B, 3D). This, together with aclearing method that does not affect the blue stain (see Protocol 6, steps 4-6),increased the contrast between stained and non-stained cells sufficientlyto permit computer-aided image analysis for the quantification of blue cells(27).

Protocol 6. Histological GUS assay of cassava tissues

Equipment and reagents• Incubator (37°C)• Slide warmer• Desiccator and vacuum pump• Assay buffer: 0.08 M sodium phosphate pH

7, 0.8 mM 5-bromo-4-chloro-3-indolyl-B-D-glucuronide cyclohexyl-ammonium salt (X-gluc; Biosynth), 0.16% Triton X-100(Sigma), and 20% (v/v) methanol

• Clearing solution (optional): 160 g chloralhydrate, 50 ml water pH 7

• Assay buffer A: for tissue from embryo-genie callus or suspensions and embryosadd 6.4 mM potassium ferrocyanide andpotassium ferricyanide

• Assay buffer B: for leaves, shoots, androots reduce the concentrations to 0.64 mMpotassium ferrocyanide and potassium fer-ricyanide

• Phenolic glycerol gelatin (Sigma)

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A. Tissue from embryogenic callus or suspensions and embryos

1. Incubate tissue in assay buffer A for 2 h at 37°C.

2. Wash the tissue several times with water.

3. Observe the tissue immediately or store in 70% ethanol.

4. If a good contrast between blue cells and the surrounding tissue isdesired, transfer the tissue to clearing solution. After three days,remove the clearing solution by washing the tissue several times in50% glycerol (v/v). After the last wash blot the tissue on filter paper toremove excess liquid.

5. Transfer the tissue to a microscope slide and place it on a slidewarmer heated to 40-45°C. Cover the tissue drop by drop with lique-fied phenolic glycerol gelatin.

6. Carefully place a coverglass on the liquid glycerol gelatin and store theslide horizontally at room temperature. Samples prepared in this wayhave been kept for more than a year without noticeable change instain intensity.

B. Tissue from plants (leaves, shoots, roots)

1. Incubate the tissue in assay buffer B for 16 h at 37°C. Vacuum infiltratethe tissue to facilitate the penetration.

2. Proceed as described in part A, steps 3 and 4. If the tissue is green, thechlorophyll can be removed after washing the tissue with water byleaving it in 70% ethanol until it becomes white. The extraction ofchlorophyll can be shortened by transfer to 100% ethanol, but in thiscase shrinkage of cells can occur.

GUS expression in cassava transformed with the uidA gene driven by the35S promoter from cauliflower mosaic virus (CaMV) as detected by the histo-logical GUS assay (see Protocol 6A) is highest in embryogenic and embryotissues (Figure 3A-F). Most of the GUS positive cells are stained dark blue.When cassava tissues from regenerated, transgenic plants are subjected to thesame GUS assay conditions, macroscopically they appear light blue (Figure 4A).However, microscopic examination reveals that some cell types stain with anintensity comparable to embryogenic and embryo cells. Without further anal-ysis one might interpret GUS expression patterns as shown in Figure 4 as celltype-specific expression of guard cells (Figure 4B) or laticifers (Figure 4D).However, by using an assay buffer containing 1/10 of the concentration offerri- and ferrocyanide (0.64 mM each), and by extending the time for assayfrom 2 h to 16 h, all cells stain blue to dark blue (Figure 4A and 4C). The con-clusion from these findings is that the outcome of a histological GUS assay

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10: Introduction of materials into living ceils

Figure 3. GUS expression in cassava cells and tissues after bombardment with a plasmidcontaining the uidA gene under the control of the CaMV 35S promoter. (A-C) Bombard-ment of embryo clumps. (A) Embryo clump, three days after bombardment. (B) Close-upof (A); single GUS-expressing cell. Several gold particles are visible. (C) Chimeric embryosix weeks after bombardment, (D-F) Bombardment of tissue derived from embryogenicsuspensions. (D) Two days after bombardment. (E) Ten days after bombardment.(F) Transgenic embryos, ten months after bombardment. Bars in (A) 3 mm; (8} 5 um; (C)2 mm; (D) 1 cm; (E) 100 um; (F) 2 mm.

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Figure 4. (A-D) GUS expression in a cassava plant transformed with the uidA gene.(A) Longitudinal sections through the shoot tip. (B) and [C} Leaf epidermis, (D) Close-upof the section on the left shown in (A), depicting a lacticifer. The intensity of the stainduring the GUS assay depends on the assay conditions. Tissues in (A (section on the left),B, and D) were subjected to a 2 h assay with 6.4 mM ferri- and ferrocyanide, tissues in(A (section on the right) and C) to a 16 h assay with 0.64 mM ferri- and ferrocyanide in theassay buffer. (E-G) Light emission in cassava tissues transformed with the luc gene.(E) Light-emitting embryogenic tissue of cassava three months after bombardment withthe luc gene. Light emission was measured for 1 min, using an intensified VIM cameraan Argus-50 image processor (Hamamatsu Photonics). (F) Regenerated transformedplantlet. (G) The same plantlet as shown in (F); light emission measured for 5 min. (H-l)Transient expression of the gfp gene in microbombarded rice tissue. (H) Tissue derivedfrom embryogenic suspension cultures three days after bombardment. The photographwas taken with a UV light source and an FITC filter set. (I) Close-up of (H); singleGFP-expressing cell. Bars in (A) 5 mm; (B, C, D) 100 (um; IE) 2 cm; (F, G) 1 cm; (H) 1 mm;(I) 30 um. Figures E-G were kindly provided by C.J.J.M. Raemakers; Figures H and I by

A. de Kochko. Figure G with permission from Kluwer Academic Publishers.

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depends very much on the assay conditions, and that different incubationtimes and oxidation catalyst concentrations should be tested before interpret-ing the assay results. This is even more important when cell-specific promotersare used to control GUS expression.

1.3.2 Other reporter genesA major disadvantage of the histological GUS assay described above is that itis usually lethal to plant tissues and therefore cannot be used for the visualselection of transgenic tissue with the purpose of further cultivation andregeneration. Other reporter genes that have been used in conjunction withparticle bombardment of plant tissues and whose expression can be detectedin vivo are the genes for firefly luciferase (luc), genes involved in anthocyaninbiosynthesis (R and Cl), and green fluorescent protein (gfp) (see Table 2).Firefly luciferase (LUC) is an enzyme that catalyses a reaction that results inthe production of visible light. Apart from being helpful for visualizing trans-formed cells it also can be used for their selection. It is known that the antibi-otics used for the selection of transgenic tissues after transformation withantibiotic resistance genes can have negative effects on the regenerationcapacity of certain transformed tissues. LUC therefore offers a selection strat-egy that avoids this problem. The detection of LUC requires only the sub-strate, luciferin, as well as ATP, Mg2+ (both present in the plant tissue), andO2. Living plant that tissues express LUC genes after exposure to luciferinproduce light at a wavelength of 560 nm which can be recorded by low lightlevel detectors (e.g. CCD cameras), and quantified by image processing sys-tems. Raemakers et al. (20) used this approach to select transgenic tissue ofcassava. In Figure 4E-G examples for the detection of LUC activity areshown. Tissue derived from embryogenic suspensions of cassava was bom-barded with a plasmid containing the firefly luciferase coding sequence con-trolled by the cauliflower mosaic virus (CaMV) 35S promoter. Repeatedselection and subculture of light-emitting tissue resulted in an enrichment oftransformed tissue (Figure 4E), from which eventually plantlets were regener-ated (Figure 4F and 4G). Despite the advantage of a marker whose expressioncan be followed in living tissue, the use of the firefly luciferase gene to identifytransformed tissue is not as widespread as the use of the GUS gene. The rea-son is the relatively high cost for the detection equipment. Recently it wasshown that a luciferase coded by a gene isolated from another organism, themarine soft coral Renilla reniformis, produces a stronger light emission, and ismore stable than the firefly luciferase (28). This luciferase has the potential ofbeing detectable with less costly equipment and might become a usefulmarker gene in the future.

Genes encoding transcriptional activators that control anthocyanin synthe-sis in maize have been used to identify transiently or stably transformed cellsby their red coloration (25). In this case neither a substrate nor special equip-ment are needed to identify transformed cells. In addition, the red stain is

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restricted to the cell expressing the introduced gene(s), i.e. there is no prob-lem of diffusion to neighbouring cells as is the case with cells expressing GUS.Limitations that restrict the use of these genes as reporter genes are the factthat their expression can depend on the presence or absence of other genes inthe anthocyanin biosynthesis pathway; as a result they may be useful only incertain plants (e.g. maize). In addition, activation of the pathway can interferewith developmental processes: constitutive expression of the R and Cl genesin sugar cane plantlets did not allow survival of anthocyanin pigmented plantstaller than 3 cm (29).

Another marker gene that can be detected in vivo is gfp, which encodes thegreen fluorescent protein (GFP) from the jellyfish Aequorea victoria. Thisprotein fluoresces green when excited by ultraviolet (395 nm) or blue light(490 nm). The green fluorescence can be observed with a UV microscopeequipped with a FITC filter set or with a hand-held ultraviolet light. Variantsof this protein with different absorption and emission spectra have beendeveloped by changing the gene coding sequence (26). In transformationexperiments with the native gfp, low expression levels of GFP were observedin plant cells due to cryptic splice sites and poor codon usage. However,recent work with codon optimized versions of the gene has shown that expres-sion levels can be achieved that are sufficient for GFP detection in planta (30).At ILTAB preliminary experiments have been performed using a mutatedversion of GFP (mutation S65T) (26) in conjunction with microbombardmentof embryogenic tissue of rice (31). Figure 4H and 4I show transient expressionof GFP, two days after bombardment.

2. Microinjection as a preparative technique formicroscopical analysisH. F. PATERSON

2.1 Advantages of microinjection by glass capillaryneedle

Although there are now numerous, widely-used methods available for intro-ducing exogenous substances into mammalian cells in culture (see Chapter 6),no technique has proved itself so well-suited or adaptable for subsequentmicroscopic analysis as direct microinjection by glass needle. Not only arethere few constraints on the molecular size or nature of the substances whichcan be introduced in this fashion, but also the intracellular site (nucleus orcytoplasm) and the quantity of the injected material can be controlled andrecorded on a cell-by-cell basis. Adherent cells growing on a tissue culturesubstratum are microinjected in situ with minimal disruption or damage,allowing unhindered observation of rapidly occurring responses to the in-

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jected material. Additionally, the ability to microinject discrete groups of cellsin adjacent areas of the culture dish with experimental and control substancesallows subsequent comparison of their effects within the same microscopicalfield.

Microinjection is applicable to all mammalian cells which grow (or can beinduced to grow) as an adherent monolayer culture, and success is far lessinfluenced by cell type than most other methods. Where substances aremicroinjected directly into the cytoplasm, efficiency of delivery may approach100%, and intracellular concentrations > 5 mg/ml of the injected substanceare obtainable if required. Efficiency of expression of microinjected DNA ismore variable, since successful introduction of plasmid into the cell nucleusdoes not guarantee gene expression. As with all methods of DNA delivery,good gene expression following microinjection is dependent on the choice of asuitable vector.

2.2 Equipment required for microinjection of adherentmammalian cells

Microinjection may be performed with relatively simple equipment. Basic re-quirements are for a microscope through which the cells and the injection pro-cedure may be observed, together with some form of micromanipulator whichallows the operator fine control over the movement of the injection needle.Access to a needle pulling device is also necessary, enabling consistently-shaped needles to be produced from capillary tubes. Finally, a source ofvariable air or hydraulic pressure is needed to force the injection solutionfrom the needle into the cell.

2.2.1 MicroscopesA compound microscope forms the basis of any microinjection system. Thisshould be equipped with at least three objective lenses, enabling the injectionneedle to be gradually manoeuvred to the centre of the field by progressiveincreases in magnification. A typical system employs X 5, X 20, and X 40objectives, in conjunction with X 10 eyepieces. Phase-contrast is generallyfound to be the most useful optical system for rendering the cells and theirinternal structures visible during the injection process, being relatively inex-pensive, straightforward to use, and robust in operation. Alternatively, DICor Nomarski optics provide unrivalled perception of depth and detail withinthe specimen, but suffer the disadvantage of being unsuitable for use withplastic tissue culture vessels.

Inverted microscopes are the most easily adapted for microinjection usage,since their design is ideally suited to working with live cells in culture dishes.When fitted with long working distance condensers, such microscopes permitunrestricted access of the injection needle into the culture dish. Conventionalupright microscopes may be used (Figure 5a), but have several shortcomings

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Figure 5, Equipment required for microinjection. (a) Basic manual system employing anupright microscope, Leitz mechanical micromanipulator (m) with integral hanging joy-stick (j), and 50 ml syringe as pressure source (s). Note 45° angle of needle holder (n). (b)Zeiss semi-automatic microinjection workstation employing an inverted microscope withincubator jacket, and Eppendorf 5170 stage-mounted electrically operated micromanipu-lator (m) with remote upright joystick (j).

by comparison with inverted types. Not only is there little room between theculture dish and the objective lens for positioning the injection needle, but themeniscus formed around the needle as it passes through the surface of the cul-ture medium, beneath the objective, and close to the optical axis, may causedegradation of the image quality. A further problem with the majority ofupright microscopes is that focusing is achieved by moving the height of thestage relative to the stationary objective turret. This design feature renders themicroscope incompatible with separately mounted micromanipulators, sinceany attempt at refocusing during microinjection will cause the cells to move inthe vertical axis relative to the needle.

An optional refinement for the microscope is the provision of a regulatedenvironment for the cells during lengthy microinjection sessions. This norm-ally takes the form of a heated stage (incorporating a suitable recess to acceptthe culture dish) surrounded by a transparent incubator jacket equipped withadjustable temperature and CO2 control, as in Figure 5b. Since a sizeable holemust be left open in the incubator jacket for access to the cells by the micro-manipulator, the control system should be of high capacity and capable ofconstantly monitoring and adjusting the internal atmosphere.

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2.2.2 MicromanipulatorsSeveral designs of micromanipulator are commercially available, employingmechanical, electrical, or hydraulic principles of operation. In most models ahand-operated joystick controls movement of the needle in the horizontalplane (x and y axes), reduced proportionately by a factor of up to 200-fold(usually adjustable). Typically, one centimetre of movement at the joystickwill cause the injection needle tip to traverse a distance roughly equivalent tothe diameter of a large adherent cell. Vertical (z axis) movement of the injec-tion needle is achieved in many systems by twisting the joystick around itsown axis, although in some designs rotation of a separate wheel is required.Sophisticated models may incorporate a feature which combines vertical andlateral movements in order to effect an axial, spearing motion of the micro-injection needle. Coarse controls are also provided to facilitate comparativelylarge, rapid movements during initial setting up and changing of needles.These will often include a simple mechanism for swinging the needle into andout of the culture medium over the high side of the dish.

Detailed discussion of different micromanipulator designs is beyond thescope of this section, but the following points are worth considering. Mechan-ical micromanipulators have the advantage of robust simplicity, but thedirectness of the linkage renders them prone to the transmission of incidentalknocks and vibrations from the operator's hand through to the needle. Withfree-standing mechanical units such as the Leitz (Figure 5a), it is also impera-tive to use a fixed-stage microscope. Hydraulically operated and electricallydriven systems allow flexible linkage, so that the needle holder and its driveunit can be mounted directly on the microscope stage while the joystick con-trols are separately sited on the base plate or table top. This arrangementkeeps the transmission of unwanted vibration to a minimum. A furtheradvantage of electrically driven systems is their potential for computerizedcontrol, permitting automatic or semi-automatic microinjection of cellsselected by the operator (Figure 5b).

2.2.3 Needle pullersA mechanized puller is required to produce consistently-shaped needles fromglass microcapillaries. Several devices are commercially available, although asimple machine can be constructed in the laboratory workshop. With the mostcommon design a straight-walled capillary tube, encircled at its mid-point by asmall electrical heating coil, is held under tension at both ends (Figure 6a).When the heating coil is made red hot, the softened length of glass in theadjacent middle section of the capillary is drawn out and eventually pulledapart into two identical sharp points. The shape of the twin needles thusformed can be controlled by altering the temperature of the heating coil andby adjusting the tension force on the ends of the capillary. Most modernmachines allow a two-stage pull to be applied to the capillary, thereby giving

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Figure 6. Needles for microinjection. (a) Central section of a Narishige PE-2 needle puller,showing close-up of capillary held under tension (c), and heating:coil (h). (b) Comparisonof capillary tubing used to make microinjection needles (c), finished needle loaded withinjection solution (n), and tip of Eppendorf microloader (ml). Note the internal filament(arrowed) in capillary and needle. Bar = 1 mm.

greater control over the dimension and taper of the needle tips. Initial gentlestretching of the heated region to a predetermined length, in order to narrowthe capillary diameter, triggers the application of a stronger secondary pullwhich forms the final taper.

2.2.4 Pressure control unitMeans of applying sufficient pressure to force the injection solution throughthe minute hole in the tip of the microcapillary needle must be provided. Theneedle is firmly gripped by an airtight rubber seal in the needle holder, whichis connected to a suitable source of air pressure via a length of small-bore,flexible, pressure tubing. For simple, non-automatetE microinjection, a largehand-held syringe provides a surprisingly capable and continuously variablepressure source (see Figure 5a). More advanced systems employ a source ofcompressed air connected to a series of valves, which allow pre-set pressuresto be delivered to the needle. The Eppendorf System 5242 incorporates threeadjustable control valves for use with automated or semi-automated systemsof microinjection. The first is designed to deliver a low, continuous pressurewhich counteracts the tendency of the needle to draw up liquid from theculture medium by capillary attraction. The second valve is operated either by

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foot switch (semi-automated systems) or computer (fully automated systems)to deliver the short pulse of higher pressure required to inject the cells. Thethird valve can be set to pass full pressure from source, and is intendedprimarily as a means of clearing blockages in the needle.

2.3 Preparation of materials for microinjection2.3.1 Cell culturesCells for microinjection may be seeded either onto plastic dishes or glasscoverslips. Standard tissue culture Petri dishes of 50-60 mm diameter are con-venient for most purposes, and adaptors to hold these dishes snugly on themicroscope stage are available from microscope manufacturers. Smallerdishes (30 mm diameter) may cause difficulty of access for the injection nee-dle. Glass coverslips must be cleaned of grease and powdered glass before useby washing in absolute alcohol. After sterilization by flaming, they are placedin Petri dishes and seeded with cells in the normal way.

Only firmly adherent cells are suitable for microinjection. Dishes pre-coated with poly-lysine, collagen, fibronectin, or laminin such as the 'BioCoat'range from Collaborative Biomedical Products will often improve the ad-hesion of loosely attached cell types.

2.3.3 Capillaries and needlesMechanical pulling devices constitute the only reliable way to create repro-ducibly-shaped microinjection needles from glass capillaries. Optimal settingsof such machines must be determined by trial and error. Capillary tubes suit-able for making needles contain a fine glass filament running along the entirelength of the inner wall (arrowed in Figure 6b) which has the important propertyof drawing the injection solution down to the very tip of the needle withoutairlocks. Needles with a taper such as shown in Figure 6b are slender enoughin use to produce minimal interference with the optics of the injection micro-scope, sufficiently rigid to be moved through the culture medium without flexing,and consistently open at the tip. The high temperature involved in pulling thecapillaries renders the needles sterile and ready for use. Protected from dust,they may be stored for several weeks until required.

Needles are loaded with injection sample immediately before use by meansof very long, fine, micropipette tips (e.g. Eppendorf 'microloaders'), sufficientlynarrow in diameter to be inserted into the shaft of the needle (see Figure 6b).

2.3.2 Marking the area of cells to be injectedSeveral million adherent cells can be accommodated on the surface of a cul-ture dish, and even a small coverslip will support many thousands. Thereforeit is important that the position of microinjected cells on the culture surfaceshould be marked so that they can be easily relocated for analysis. When plasticPetri dishes are used, this can be done after cells have been plated. A region

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Figure 7. Microinjection techniques, (a) Injection into the nucleus of an epithelial cell,showing the phase-bright halo around the tip of the needle (arrowed). Bar = 30 um. (b) Asingle field of mouse fibroblasts divided into four quadrants by scoring a cross in the sur-face of the culture dish. The cells in each quadrant have been microinjected with differentrecornbinant ras proteins, allowing direct comparison of their effect on morphology.Bar = 200 um.

of the dish should be selected where the cells are evenly distributed and freeof clumps or other anomalies. With the aid of a low power inverted micro-scope, two lines forming a cross are scored on the upper (tissue culture) sur-face of the dish using a sterile scalpel blade, thereby providing four adjacentquadrants of cells for injection (Figure 7b). More elaborate box or gridpatterns may be fashioned according to the demands and complexity of theexperiment. This technique has the advantage of ensuring that the markinglines are in the same focal plane as the cells, permitting both to be resolvedsimultaneously under high magnification where depth of field is very limited.The ridge of plastic thrown upwards by the scalpel blade also constitutes auseful physical barrier to cell movement between adjacent marked areas.

Marking coverslips by hand is less precise, since a diamond-point scriber orsimilar instrument must be used to scratch suitable markings on the glasssurface prior to seeding the cells. More satisfactory are the pre-sterilized'Cellocate' coverslips from Eppendorf which are embossed in the centre witha labelled grid to allow a wide choice of well-defined, easily relocated areas ofcells for microinjection.

2.4 Microinjection techniqueA step by step procedure for microinjecting cells is given in Protocol 7, modi-fied from the technique of Graessmann and Graessmann (32). The descrip-tion is confined to manual injection which can be used with the most simple

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equipment. Those intending to employ automated systems should follow theguide-lines provided by the manufacturer.

In this basic form of microinjection the tip of the loaded needle is main-tained at an angle of 45 degrees to the surface of the culture dish, and islowered vertically into the cell with a slicing movement similar to cutting acake. The needle contents, which are kept under sufficient air pressure tomaintain a continuous stream from the tip, will begin to flow into the cell. Thisis evidenced by swelling of the injected compartment, and movement of intra-cellular structures (Figure 7d). Experience will guide the operator in judgingwhen to raise the needle out of the cell in order to complete the injectionprocess. Under phase-contrast optics any cells which have been killed byover-injection become evident within a few minutes, exhibiting dark shrunkennuclei and coarsely speckled cytoplasm. For most cells, injection into the cyto-plasm is better tolerated than into the nucleus. No fixed figures can be givenconcerning injection pressure or duration, as this is dependent on the apertureof the needle tip, the viscosity of the injection solution, and the type of cell.However, for good viability the pressure should be high enough to keep injec-tion duration to less than half a second. Many cell types will survive injectionof up to 10% of their own volume, although smaller quantities are desirable toavoid unwanted stress. Graessmann and Graessmann (32) have determinedmean injection volume to be 1-2 X 10~u ml/cell. Up to 500 cells per hour maybe microinjected using this technique.

Protocol 7. Technique for manual microinjection of adherentmammalian cells in culture

Equipment and reagents• Compound microscope equipped with x 5, • Needles pulled from glass capillaries with

x 20, and x 40 objectives and phase-con- internal filament, 100 mm long x 1.2 mmtrast optics external diameter x 0.69 mm internal di-

• Micromanipulator, with 1.2 mm gauge ameter (GC120F-10, Clarke Electromedicalneedle holder Instruments)

. Pressure source: 50 ml syringe, or com- • Injection sample, prepared in Dulbecco'spressed air with pressure control unit phosphate-buffered saline (PBSa)

• Adjustable micropipette and 'microloader' • Adherent cells cultured in marked 50 or 60tips (Eppendorf) mm diameter Petri dishes

Method

1. Place the prepared Petri dish, containing normal culture medium, onthe microscope stage. Locate the marked area of cells to be injected,and bring into focus with the x 5 phase-contrast objective.

2. Draw 2-3 ul of injection sample solution into a microloader, and insertcarefully into the shaft of a microinjection needle from the back-end.Dispense 0.5-1 ul of the sample into the tapered end of the needle,ignoring any bubbles which may form.

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Protocol 7. Continued3. Insert the rear half of the loaded needle into the needle holder, and

tighten the securing ring to ensure a firm, airtight seal. Clamp theassembly into the micromanipulator, ensuring that the needle pointsdownwards at an angle of 45° to the surface of the culture dish (seeFigure 5a).

4. Use the coarse controls of the micromanipulator to swing the needleover the side of the dish, and guide it towards the central, illuminatedarea.

5. Lower the needle into the culture medium. Using the X 5, x 20, and x 40objectives sequentially, bring the needle tip to the exact centre of themicroscope field, resting in partial focus above the cell monolayer.Reset the joystick to the centre of its travel.

6. Deliver a burst of high pressure to the needle in order to initiate theflow of injection solution. Adjust the pressure to give light continuousflow (~ 100-400 hPa on a pressure regulator, or light finger pressureon a 50 ml syringe).

7. Select a group of cells for practice, just outside the marked experimentalarea, and position the needle directly above a test cell by means of thejoystick. Lower the tip vertically until the presence of a small, phase-bright halo indicates contact with the plasma membrane (Figure 7a).Lower the tip further until the cytoplasm or nucleus is penetrated,hesitate until a slight swelling reveals that injection has occurred, thenswiftly withdraw the needle up to the resting position.

8. Examine the cell for signs of over-injection (see text), and adjust theneedle pressure accordingly. Perform further test injections, on adjacentcells, until satisfied with viability.

9. Return to the marked area, and microinject as many cells as requiredfor the experiment.

2.5 Microscopical analysis of microinjected cellsAnalysis of microinjection experiments is almost invariably performed micro-scopically, since the number of injected cells rarely constitutes sufficient mate-rial for biochemical techniques. In most cases groups of microinjected cellsare examined either by conventional or time lapse photomicroscopy forchanges in behaviour and morphology, or fixed and analysed using standardimmunofluorescence protocols with antibodies against injection-derived orassociated endogenous proteins. Where antibodies are not available againstthe injected material, it is often helpful to incorporate a suitable inert markerinto the injection solution, such as 1 mg/ml purified immunoglobulin, which isretained within the cell and can be stained to identify injected cells during

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Figure 8. Analysis by fluorescence microscopy in living cells of the intracellular localiza-tion of raf-GFP fusion protein, 16 h after microinjection of DNA vector. (a) Live cellsexpressing raf-GFP alone exhibit cytosolic localization of the protein, (b) Co-expressionwith oncogenic ras (not visible) causes translocation of raf-GFP to the plasma mem-brane. x 40 water immersion objective with standard FITC fluorescence filter set. Bar =20 um.

subsequent analysis. An important technique for analysis is that of 'epitopctagging', where the DNA coding sequence of a gene is modified such that theprotein expressed contains an additional small polypeptide 'tag' correspond-ing to the binding site of a pre-existing, high affinity antibody. Immunostain-ing is performed against the tag, obviating the need to raise and characterizeantibodies specific for the original protein. An additional advantage is that thelocalization of exogenous proteins containing experimental mutations can beanalysed in isolation from the endogenous wild-type pool (33). A recentdevelopment in protein tagging has been the discovery of green fluorescentprotein (GFP), an intrinsically fluorescent polypeptide, variants of which canbe detected with fluorescence filter sets routinely used for fluorescein (FITC)immunostaining. When the DNA coding sequence for GFP is suitably incor-porated into a gene of interest, and introduced into cells via an appropriateexpression vector, the resultant fused protein can be detected in vivo by fluor-eseenee imaging as shown in Figure 8. Using CCD video fluorescence micro-scopy, the intracellular localization and movements of GFP fusion proteinsexpressed from microinjected plasmids can be observed and recorded in livingcells over extended periods of time (34),

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AcknowledgementsThe authors (C. S. and C. M. F.) wish to thank Dr R. N. Beachy (The ScrippsResearch Institute, La Jolla, USA) for his critical review of their manuscript.

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12. Reiser, W. (1992). Optimisation of biolistic transformation using the helium-drivenPDS-1000-He system. Bio-Rad Bulletin 1688, Bio-Rad Life Science Group.

13. Sanford, J.C., DeVit, M.J., Wolf, E.D., Russel, J.A., Smith, F.D., Harpending,P.R., et al. (1991). Technique, 3, 3.

14. Southgate, E.M., Davey, M.R., Power, J.B., and Marchant, R. (1995). Biotechnol.Adv., 13, 631.

15. Birch, R.G. and Bower, R. (1994). In Particle bombardment technology for genetransfer (ed. N.-S. Yang and P. Christou), p. 3. Oxford University Press, NewYork.

16. Sivamani, E., Shen, P., Opalka, N., Beachy, R.N., and Fauquet, C.M. (1996). PlantCell Rep., 15, 322.

17. Russell, D.A. and Fromm, M.E. (1995). In Gene transfer to plants (ed. I. Potrykusand G. Spangenberg), p. 118. Springer-Verlag, Berlin, New York.

18. Schopke, C., Chavarriaga, P., Mathews, H., Li, G.-G., Fauquet, C., and Beachy,R.N. (1993). In Vitro Cell. Dev. Biol., 29A, 64A.

19. Schopke, C., Taylor, N., Carcamo, R., Konan, N.K., Marmey, P., Henshaw, G.G.,et al. (1996). Nature Biotechnol., 14, 731.

20. Raemakers, C.J.J.M., Sofiari, E., Taylor, N., Henshaw, G., Jacobsen, E., andVisser, R.G.F. (1996). Mol. Breeding, 2, 339.

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21. Szabados, L., Hoyos, R., and Roca, W.M. (1987). Plant Cell Rep., 6, 248.22. Raemakers, C.J.J.M., Jacobsen, E., and Visser, R.G.F. (1995). In The cassava

biotechnology network; proceedings of the second international scientific meeting,Bogor, Indonesia, 22-26 August 1994 (ed. W.M. Roca and A.M. Thro), Vol. I,p. 336. CIAT, Cali, Colombia.

23. Taylor, N.J., Edwards, M., Kiernan, R.J., Davey, C., Blakesley, D., and Henshaw,G.G. (1996). Nature Biotechnol, 14, 726.

24. Jefferson, R.A. (1987). Plant Mol. Biol. Rep., 5, 387.25. Bowen, B. (1992). In GUS protocols: using the GUS gene as a reporter of gene

expression (ed. S.R. Gallagher), p. 163. Academic Press, San Diego.26. Cubitt, A.B., Heim, R., Adams, S.R., Boyd, A.E., Gross, L.A., and Tsien, R.Y.

(1995). Trends Biochem. Sci, 20, 448.27. Schopke, C., Taylor, N.J., Carcamo, R., Beachy, R.N., and Fauquet, C. (1997).

Plant Cell Rep., 16, 526.28. Mayerhofer, R., Langridge, W.H.R., Cormier, M.J., and Szalay, A.A. (1995). Plant

J.,7, 1031.29. Bower, R., Elliott, A.R., Potier, B.A.M., and Birch, R.G. (1996). Mol. Breeding, 2,

239.30. Leffel, S.M., Mabon, S.A., and Stewart, C.N. (1997). Biotechniques, 23, 912.31. Zhang, S., Chen, L., Qu, R., Marmey, P., Beachy, R.N., and Fauquet, C.M. (1996).

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Wu, L. Grossman, and K. Moldave), Vol. 101, p. 482. Academic Press, London.33. Paterson, H., Adamson, P., and Robertson, D. (1995). In Methods in enzymology

(ed. W. Balch, C. Der, and A.Hall), Vol. 256, p. 162. Academic Press, London.34. Kaether, G. and Gerds, H.-H. (1995). FEBS Lett., 369, 267.

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Surface fluorescence microscopywith evanescent illumination

D.AXELROD

1. Fluorescence at surfacesThe interaction of molecules with surfaces is central to numerous phenomenain biology: e.g. binding to and triggering of cells by hormones, neurotransmit-ters, and antigens; the deposition of proteins upon foreign surfaces leading tothrombogenesis; electron transport in the mitochondrial membrane; celladhesion to surfaces; concentration of reactants at surfaces and consequentenhancement of their reaction rates with other surface bound molecules; thedynamical arrangement of submembrane cytoskeletal structures involved incell shape, motility, and mechanoelastic properties; and molecular eventspreceding cellular secretion from intracellular granules.

In most of these examples, certain functionally relevant molecules coexistin both a surface-associated and non-associated state. If such molecules aredetected by a conventional fluorescence technique (such as epi-illumination ina microscope), the fluorescence from surface-associated molecules may bedwarfed by the fluorescence from non-associated molecules in the adjacent de-tection volume. As an optical technique designed to overcome this problem,total internal reflection fluorescence (TIRF) allows selective excitation of justthose fluorescent molecules in close (~ 100 nm) proximity to the surface.

One note of caution: TIRF generally involves the use of a laser (with greenor blue colour) for fluorescence excitation. Appropriate care should be takento protect the eyes against exposure to direct or reflected laser light by the useof attenuating goggles when possible.

1.1 TIRF for biochemical samplesTIRF can be used quantitatively on non-microscopic or featureless samples tomeasure concentrations of fluorophores as a function of distance from thesubstrate, or to measure binding/unbinding equilibria and kinetic rates at abiological surface. This type of study can be done with a custom TIRF con-figuration in a commercial spectrofluorimeter, but it is just as convenient and

11

D. AxeIrod

Figure 1. Three views of a mouse BC3H1 smooth muscle cell micro injected with rhodaminedextran: (a) TIR, using configuration E (Section 3.3); (b) epi-illumination; and {c} phase-contrast. The objective was a Zeias x 40 water immersion, NA - 0.75 achromat, used ona Leitz Diavert microscope. The images were recorded digitally on a 576 l. 384 pixel slowscan cooled CCD camera ( S t a r , Photometries Inc., Tucson, AZ), The image was thendisplayed on a VGA monitor screen using custom FORTRAN-based software and photo-graphed onto standard 35 mm film.

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11: Surface fluorescence microscopy with evanescent illumination

much more light-efficient to use a microscope adapted for TIRF. Recently,single molecule fluorescence in biochemical systems has been detected byreducing extraneous fluorescence from out-of-focus planes, notably by TIRF.

1.2 TIRF for biological samplesAs applied to biological cell cultures observed through a microscope, TIRFallows selective visualization of cell/substrate contact regions, even in samplesin which fluorescence elsewhere would otherwise obscure the fluorescentpattern in contact regions. TIRF can be used qualitatively to observe theposition, extent, composition, and motion of these contact regions. Figure 1shows an example of TIRF on a intact living cells in culture, compared withstandard epifluorescence and phase-contrast.

Since the cells are exposed to excitation light only at their cell/substratecontact regions but not through their bulk, they tend to survive longer underobservation. This makes TIRF time lapse movies quite feasible, even overperiods of days. TIRF is easy to set up on a conventional upright or invertedmicroscope with a laser light source or, in a special configuration, with a con-ventional arc source. TIRF is completely compatible with standard epifluor-escence, bright-field, dark-field, or phase-contrast illumination; these methodsof illumination can be switched back and forth readily. Since the illuminationsystem can be 'chopped' between TIRF and standard through the lens 'epi'illumination, fluorescence changes at the submembrane and deeper in thecytoplasm can be simultaneously recorded and compared.

2. Theory of TIRFTIRF is conceptually simple. An excitation light beam travelling in a solid (e.g.a glass coverslip or tissue culture plastic) is incident at a high angle 0 upon thesolid/liquid surface to which the cells adhere. That angle 6, measured from thenormal, must be large enough for the beam to totally internally reflect ratherthan refract through the interface, a condition that occurs above some 'criticalangle'. TIR generates a very thin (generally less than 200 nm) electromagneticfield in the liquid with the same frequency as the incident light, exponentiallydecaying in intensity with distance from the surface. This field is capable ofexciting fluorophores near the surface while avoiding excitation of a possiblymuch larger number of fluorophores farther out in the liquid.

2.1 Single interfaces: intensity and polarization2.1.1 Infinite incident plane wavesWhen a light beam propagating through a transparent medium 3 of high indexof refraction (e.g. glass) encounters a planar interface with medium 1 of lowerindex of refraction (e.g. water), it undergoes total internal reflection for

401

where n1 and n3 are the refractive indices of the liquid and the solid respec-tively, and n - n1/n3. Ratio n must be less than unity for TIR to occur. (Arefractive index n2 will refer to an optional intermediate layer to be discussedin Section 2.2.) For incidence angle 0 < 0C, most of the light propagatesthrough the interface with a refraction angle (also measured from the normal)given by Snell's Law. (Some of the incident light internally reflects back intothe solid.) For 0 > 0C, all of the light reflects back into the solid. However,even with TIR, some of the incident energy penetrates through the interfaceand propagates parallel to the surface in the plane of incidence. The field inthe liquid, called the 'evanescent field' (or 'wave'), is capable of exciting fluo-rescent molecules that might be present near the surface.

For an infinitely wide beam (i.e. a beam width many times the wavelengthof the light, which is a good approximation for unfocused or weakly focusedlight), the intensity of the evanescent wave (measured in units of energy/area/sec) exponentially decays with perpendicular distance z from the interface:

l0 is the wavelength of the incident light in vacuum. Depth d is independentof the polarization of the incident light and decreases with increasing 0.Except for 0 —> 0C (where d -» °°), d is in the order of l0 or smaller. A morecomplete mathematical description of the electric and magnetic fields of theevanescent wave produced by infinite incident plane waves can be found inref. 1.

A physical picture of refraction at an interface shows TIR to be part of acontinuum, rather than a sudden new phenomenon appearing at 6 = 0C. Forsmall 0, the refracted light waves in the liquid are sinusoidal, with a certaincharacteristic period noted as one moves normally away from the surface. As0 approaches 0C, that period becomes longer as the refracted rays propagateincreasingly parallel to the surface. At exactly 0 = 0C, that period is infinite,since the wavefronts of the refracted light are themselves normal to thesurface. This situation corresponds to d = °°. As 0 increases beyond 0C , theperiod becomes mathematically imaginary; physically, this corresponds tothe exponential decay of Equation 2.

The polarization of the electric field of the evanescent wave depends on theincident light polarization, which can be either 's' (polarized normal to theplane of incidence formed by the incident and reflected rays) or 'p' (polarized in

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D. Axelrod

incidence angles (measured from the normal to the interface) greater than the'critical angle'. The critical angle 0C for TIR is given by:


Figure 2. Polarization snapshot of the electric field for p-polarized (in-plane arrows) ands-polarized (dots) light at a TIR surface. 8 refers to the non-zero phase shift between theincident light at the TIR surface and the evanescent wave.

the plane of incidence). For s-polarized incident light, the evanescent electricfield vector direction remains purely normal to the plane of incidence. For p-polarized incident light, the evanescent electric field vector direction remainsin the plane of incidence, but it 'cartwheels' along the surface with a non-zerolongitudinal component (see Figure 2). This feature distinguishes evanescentlight from freely propagating subcritical refracted light, which has no longi-tudinal component. The longitudinal component approaches zero as theincidence angle is reduced from the supercritical range back toward the criticalangle.

Regardless of polarization, the spatial period of the evanescent electric fieldis lo/(n3 sin 0) as it propagates along the surface. Unlike the case of freelypropagating light, the evanescent spatial period is not at all affected by themedium 1 in which it resides. It is determined only by the spacing of the inci-dent light wavefronts in medium 3 as they intersect the interface.

The product of the evanescent electric field E with its complex-conjugate E*is proportional to the probability rate of energy absorption by a fluorophorein the evanescent wave. This product is the 'intensity' I in Equation 2. Givencorresponding incident intensities I'p,s, the evanescent intensities Ip>s at z = 0are:

Intensities Ip,S(0) are plotted versus 0 in Figure 3, assuming the incidentintensities in the glass I'p>s are set equal to unity. The evanescent intensityapproaches zero as 0 -» 90°. On the other hand, for supercritical angles withinten degrees of 6C, the evanescent intensity is as great or greater than theincident light intensity. The plots can be extended without breaks to the

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D. Axelrod

Figure 3. Intensity EE" (proportional to the probability of excitation of a fluorophore) versusincidence angle 6 for transmitted light in the low refractive index medium 1 at z = 0 at aTIR interface. The incident intensity in medium 3 is assumed to be unity. At angles 0 > 6C,the transmitted light is evanescent; at angles 0 < 0C, it is propagating. Both s- and p-polar-izations are shown. Refractive indices n3 = 1.46 (fused silica) and n1 = 1.33 are assumedhere, corresponding to 0C = 65.7°.

subcritical angle range, where the intensity is that of the freely propagating re-fracted light in medium 1. One might at first expect the subcritical intensity tobe slightly less than the incident intensity (accounting for some reflection atthe interface) but certainly not more as shown. The discrepancy arises be-cause the intensity in Figure 3 refers to EE* alone rather than to the actualenergy flux of the light, which involves a product of EE* with the refractiveindex of the medium in which the light propagates.

2.1.2 Finite width incident beamsFor a finite width beam, the evanescent wave can be pictured as the beam'spartial emergence from the solid into the liquid, travel for some finite distancealong the surface, and then re-entrance into the solid. The distance of propa-gation along the surface is measurable for a finite width beam and is called theGoos-Hanchen shift. The Goos-Hanchen shift ranges from a fraction of awavelength at 0 = 90° to infinite at 0 = 6C , which of course corresponds to therefracted beam skimming along the interface. A finite beam can be expressedas an integral of infinite plane waves approaching at a range of incidenceangles. In general, the intensity profile of the finite beam evanescent field canbe calculated from the mathematical form for the evanescent wave at each in-finite plane wave incidence angle, integrated over all the constituent incidentplane wave angles.

404


Figure 4, Intensity profile of the TIR region illuminated by a Gaussian profile beam from aCW argon laser, as visualized upon a Dil coated surface (see Protocol 1). Configuration A(see Section 3.3) was used here with a Leitz X 10, NA = 0.25 air achromat objective on aLeitz Diavert microscope, (a) Beam slightly defocused at the TIR surface, (b) Beamfocused at the TIR surface. The incidence angle is about 2° greater than the critical angle.

For a TIR Gaussian laser beam focused with a typically narrow angle ofconvergence, the experimentally observed evanescent illumination is approx-imately an elliptical Gaussian profile, and the polarization and penetrationdepth are approximately equal to those of a single inf in i te plane wave. How-ever, if the angle of convergence is greater and the mean angle is within afew degrees of the critical angle, the evanescent field tends to become a longthin stripe (see Figure 4). A more complete mathematical description ofthe evanescent wave produced by a focused TIR laser beam can be found inref. 2.

2.2 Intermediate dielectric layersIn actual experiments in biophysics, the interface may not be a simple inter-face between two media, but rather a stratified multilayer system. One exampleis the case of a biological membrane or lipid bilayer interposed between glassand an aqueous medium. Another example is a thin metal film coating, whichcan be used to quench fluorescence within the first — 10 nm of the surface.We discuss here the TIR evanescent wave in a three layer system in whichincident light travels from medium 3 (refractive index n3) through the inter-mediate layer (n2) toward medium 1 (n1).

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D. Axelrod

Qualitatively, several features can be noted:

(a) Insertion of an intermediate layer never thwarts TIR, regardless of theintermediate layer's refractive index n2. The only question is whether TIRtakes place at the n3:n2 interface or the n2:n1 interface. Since the inter-mediate layer is likely to be very thin (no deeper than several tens ofnanometres) in many applications, precisely which interface supports TIRis not important for qualitative studies.

(b) Regardless of n2 and the thickness of the intermediate layer, the evanes-cent wave's profile in medium 1 will be exponentially decaying with acharacteristic decay distance given by Equation 3. However, the overalldistance of penetration of the field measured from the surface of medium3 is affected by the intermediate layer.

(c) Irregularities in the intermediate layer can cause scattering of incidentlight which then propagates in all directions in medium 1. Experimentally,scattering appears not be a problem on samples even as inhomogeneousas biological cells. Direct viewing of incident light scattered by a cell sur-face lying between the glass substrate and an aqueous medium confirmsthat scattering is many orders of magnitude dimmer than the incidentor evanescent intensity, and will thereby excite a correspondingly dimcontribution to the fluorescence.

2.3 Intermediate metal filmA particularly interesting kind of intermediate layer is a metal film. Theory(1) shows that such a film will reduce the s-polarized evanescent intensity tonearly zero at all incidence angles. But the p-polarized behaviour is quitedifferent. At a certain sharply defined angle of incidence 6P ('the surfaceplasmon angle'), the p-polarized evanescent intensity becomes an order ofmagnitude brighter than the incident light at the peak. This strongly peakedeffect is due to a resonant excitation of electron oscillations at the metal/waterinterface. For an aluminium film at a glass/water interface, 6p is greater thanthe critical angle 0C for TIR. The intensity enhancement is rather remarkablesince a 20 nm thick metal film is almost opaque to the eye.

There are some potentially useful experimental consequences of TIRexcitation through a thin metal film coated on glass:

(a) The metal film will almost totally quench fluorescence within the first10 nm of the surface, and the quenching effect is virtually gone at a distanceof 100 nm. Therefore, TIR with a metal film coated glass can be used toselectively excite fluorophores in the 10-200 nm distance range.

(b) A light beam incident upon a 20 nm aluminium film from the glass sideat a glass/aluminum film/water interface evidently does not have to becollimated to produce TIR. Those rays that are incident at the surfaceplasmon angle will create a strong evanescent wave; those rays that are

406


too low or high in incidence angle will create a negligible field in thewater. This phenomenon may ease the practical requirement for a colli-mated incident beam in TIR and make it easier to set up TIR with aconventional arc light source.

(c) Thirdly, the metal film leads to a highly polarized evanescent wave (pro-vided I'p = 0), regardless of the purity of the incident polarization.

3. Optical configurationsA wide range of optical arrangements for TIRF have been employed. In general,an inverted microscope is more convenient because it provides more room toadd TIR optics above rather than below the stage. However, some uprightmicroscope configurations are still very workable. Most configurations use anadded prism to direct the light toward the TIR interface, but it is also possibleto use the microscope objective itself for this purpose. This section givesexamples of these arrangements, partly as a guide to simple systems that workand partly as a basis for creative variations. For concreteness in the descrip-tions, we assume that the sample consists of fluorescence labelled cells inculture adhered to a glass coverslip.

In all cases, the critical angle must be considered in the design. The index ofrefraction of the standard glass coverslip upon which cells are grown is aboutn1 = 1.52. The index of refraction of the intact cell interior can be as high asn3 = 1.38. Therefore, to obtain TIR at this interface, the angle of incidencemust be larger than the critical angle of 65°. If the cells are not intact (e.g. per-meabilized, haemolysed, or fixed) so that the lower refractive index is that ofaqueous buffer (n1 = 1.33) instead of cytoplasm, then the critical incidenceangle is 61°.

Alignment of the optics for TIR is not difficult but it is not immediatelyobvious either. Immediately after the sections for prism-based TIR and forprismless TIR, protocol boxes are provided to help in the alignment procedure.

3.1 Inverted microscope TIR with prism on topFigure 5 shows several schematic drawings designated A-D for setting upTIR in an inverted microscope with a prism over the sample, all using a laser(usually blue or green output) as a light source. In all these configurations, thebuffer-filled sample chamber consists of a lower bare glass coverslip, a spacerring (often made of 60 um thick Teflon), and the cell coverslip inverted so thecells face down. The upper surface of the cell coverslip is put in optical contactwith the prism lowered from above by a layer of immersion oil or glycerol.The lateral prism of the prism is fixed but the sample can be translated whilestill maintaining optical contact. The lower coverslip can be oversized and theTeflon spacer can be cut with gaps so that solutions can be changed by capil-lary action with entrance and exit ports. Alternatively, commercial solution

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D. Axelrod

Figure 5, Four configurations (A-D) for an inverted microscope with a TIR prism abovethe sample. The sample is a coverslip sandwich with an intervening space; all the verticaldistances are exaggerated for clarity here. In configuration B, the dotted lines refer to theoption of truncating the prism in order to clear the optical path for a condenser system. Inconfiguration D, the option of producing interference fringes with intersecting beams isdepicted with a dashed line.

changing chambers can be used, such as the Dvorak-Stotler (Nicholson PrecisionScientific, Gailhersburg, MD), the Sykes-Moore (Bellco Glass Co. Vineland,NJ), or rectangular cross-section microcapillary tubes (Wilmad Glass, Buena.NJ).

The configurations in this set share some advantages:

(a) Inexpensive optics.(b) Ample room for set-up,(c) Prism may be mounted on condenser holder for case in raising and lowering,(d) Ease in checking alignment.

However, these configurations share some common drawbacks:

(a) Sample not easily accessible from above.(b) Shortest working distance objectives may not reach focus across the

buffer layer,(c) Image quality with highest aperture objectives somewhat reduced by-

viewing through the buffer layer.

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The four configurations A-D each have their own advantages and dis-advantages.

3.1.1 Configuration AThe rectangular solid prism allows transmitted light from the microscope con-denser above to reach the sample undistorted, so that phase-contrast anddark-field can be viewed simultaneously. Such a rectangular solid prism mustbe custom cut and polished, but can be substituted with a commercial polar-izing cube. The size of the TIR illuminated region and the incidence angle areeasily varied by adjusting the position and angle of the incoming laser beamand the focusing lens.

3.1.2 Configuration BThe triangular prism is commercially available inexpensively and in numeroussizes, generally with 45-45-90 right angle and 60-60-60 equilateral shapes.The maximum incidence angle is obtained by introducing the beam from thehorizontal direction. For standard glass (n = 1.52), the maximum incidenceangle is 73° for the right angle prism and 79° for the equilateral prism. Phase-contrast and dark-field are not compatible with this configuration because theupper surface of the triangular prism is not flat. However, custom truncationand polishing of the top of the prism (shown as an option in the figure) pro-vides compatibility with phase-contrast and dark-field.

3.1.3 Configuration CThe 60° trapezoidal prism is the most convenient and reproducible system ofall when mounted on the microscope condenser mount. The incoming beam isvertical so the TIR spot shifts laterally very little when the prism is raised andrelowered during changes of sample. Phase-contrast and dark-field are com-patible with this flat-topped prism. The incidence angle is fixed at 60°. As dis-cussed above, this angle is not sufficient to produce TIR if the prism is madefrom standard n3 = 1.52 glass. Therefore, the prism must be made of higherthan standard refractive index material (e.g. flint glass at n = 1.64). The beamwill then refract away from the normal to 0 = 69° in passing from the prisminto the coverslip, thereby exceeding the critical angle at the coverslip/wateror coverslip/cell interface. Ideally, a trapezoid with walls between 45 and 60degrees would be best, but that might have to be fabricated as a more expen-sive special order. Neither 45 or 60 degree trapezoids are commercially avail-able either, but they can be cheaply manufactured by truncating and polishingthe apex of a commercially available triangular prism (available from RolynOptics, Covina, CA in equilateral flint glass).

3.1.4 Configuration DThe parabolic mirror and hemispherical prism here are positioned so that thebeam traverses a radius of the prism toward a TIR spot at the focus of the

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Figure 6. Configuration E for an inverted microscope with a TIR prism below the sample.The lower coverslip (shown darker) is fixed by optical glue to the prism. That coverslip isoptional, intended to avoid smearing immersion oil.

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D. Axelrod

parabola. In this manner, a lateral shift of the vertical incoming beam willalways focus at the same spot. Substantial changes of incident angle therebycan be accessed quite conveniently. In addition, interference fringes in theTIR evanescent field can be created by splitting the incoming beam into twobeams, each reflecting at different azimuthal positions in the parabola butrecombining at the same parabola focus. The spacing of the fringes can beadjusted by varying the relative azimuthal positions of the two beams. Despiteall this versatility, this set-up is rather difficult to align and it can be rathersensitive to vibrations.

3.2 Inverted microscope TIR with prism belowTIR can be set up to be compatible with simultaneous microinjection or micro-electrophysiology. To provide for easy and continuous access to a liquid-containing sample from above, a prism TIR set-up should have the prismdeployed below stage level. Unfortunately, that is also where the objectiveresides, so the geometry is rather tight. Configuration E (shown in Figure 6)accomplishes the goal by taking advantage of multiple TIR in the coverslip.This transfers the excitation light as a waveguide from far off-axis to thecentre of the field of view.

In the simplest form, only a small commercially available triangular prismneed be placed in optical contact (via oil) with the bottom of the cell-containingcoverslip. The sample can be translated while the prism remains laterallyfixed. The problem is that this may leave a smear of oil on the lower side ofthe coverslip which destroys the first internal reflection. A very feasiblealternative is to use an additional intervening coverslip fixed to the prism withoptical glue. The sliding motion occurs between the intervening coverslip


and the cell coverslip, lubricated and optically contacted by a thin layer ofimmersion oil.

The disadvantage of this set-up is that oil or glycerol immersion objectivescannot be used because that might destroy the TIR of the final internal reflec-tion before the spot under view. However, water (or air) immersion objectiveswork well with this configuration.

3.3 Upright microscope TIR with prism belowPossibly the most convenient TIR set-up of all is configuration F, shown inFigure 7. The same kind of trapezoidal prism as discussed in Section 3.2 can beused here, mounted on the microscope's condenser holder. The laser beam isintroduced in the same port in the microscope base as intended for the trans-mitted light illuminator (which should be removed), thereby utilizing the

Figure 7. Configuration F for an upright microscope with a trapezoidal prism below thesample. It is shown here with a special sealed sample chamber for long-term viewing ofcells in a plastic tissue culture dish.

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D. Axelrod

microscope's own in-base optics to direct the beam vertically upward. Anextra lens just above the microscope base may be useful to position and focusthe TIR spot. As discussed earlier, flexibility in incidence angle is sacrificedfor convenience; however, a set of various angled trapezoids will allow one toemploy various discrete incidence angles.

This system gives particularly high quality images if a water immersionobjective is employed and submerged directly into the buffer solution an un-covered cell chamber. This system is also easily used with cells adhering directlyon tissue culture plastic dishes rather than on coverslips; the plastic/cell inter-face is then the site of TIR. If the objective has a long enough working dis-tance, reasonable accessibility to micropipettes is possible.

Protocol 1. Alignment of prism TIR

Equipment• Laser with blue or green output, at least • Prism as selected for the desired con-

1 W total power figuration. Fluorescence microscope equipped with • Assorted optical mounts

epi-illumination dichroic mirror and barrier • Focusing lens of approx. +50 to 150 mmfilter appropriate for the laser colour, but focal lengthwith the excitation filter removed • Safety goggles

Method

1. Mount the prism on the condenser mount carrier if possible. This neednot be done in a precision fashion, but only accurate enough so ausable area at the sample-contacting surface of the prism lies directlyin the optical axis of the microscope objective. The mounting mayrequire some custom machining of plexiglass or brass plates and useof a glue (e.g. Duco cement) that can be easily cracked off and regluedif necessary. If a standard condenser will be used for simultaneousphase-contrast or dark-field and the condenser mount cannot hold twoseparate carriers, then the prism must be mounted on a separateholder with the capability of vertical motion. If the microscope focusesby moving the stage up and down, then this separate holder must befixed to the microscope stage itself. Otherwise, the prism holder canbe fixed directly to the optical table.

2. Depending on the configuration, a system of mirrors with adjustableangle mounts fixed to the table must be used to direct the beamtoward the prism. One of these mirrors (or a system of shutters)should be movable and placed near the microscope so switchingbetween standard epi-illumination and TIR is possible without inter-rupting viewing. Mounts for a focusing lens should also be prepared.

3. Place a uniform fluorescent sample coverslip in the same kind ofsample holder to be used for cell experiments. A convenient and

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durable uniform film is made from 3,3' dioctadecylindocarbocyanine(also known as 'Dil', available from Molecular Probes, Eugene, OR). IfDil is to be used, dissolve it at about 0.5 mg/ml in ethanol, and place asingle droplet of the solution on a glass coverslip. Then, before it dries,rinse off the coverslip with distilled water. A monolayer of fluorophorewill remain adhered to the glass; the monolayer is fluorescent andstable either in air or in water. Filter sets appropriate for either fluor-escein or rhodamine will work with Dil.

4. With the uniform sample on the stage, focus on the fluorescent sur-face with transmitted (usually tungsten) illumination. Usually, dustand defects can be seen well enough to assay the focus. However, adeliberately scribed scratch in the fluorescent surface can be made toaid this focusing process. Fluorescent epi-illumination can also beused to find the focus because only at the focal position are laser inter-ference fringes seen sharply.

5. Place a small droplet of immersion oil on the non-Dil surface of thesample coverslip or directly on the prism (depending on which onefaces upward in the chosen configuration) and carefully translate theprism vertically so it touches and spreads the oil but does not squeezeit so tightly that lateral sliding motion is inhibited. Too much oil willbead up around the edges of the prism and possibly interfere with theillumination path.

6. By naked eye (perhaps with safety goggles to attenuate errant reflec-tions) and without any focusing lens in place, adjust the unfocused('raw') collimated laser beam position with the mirrors so that TIRoccurs directly in line with the objective's optical axis. This can usuallybe seen by observing the scattering of the laser light as it traversesthrough the prism, oil, and TIR surface.

7. Insert the focusing lens so that the focus is roughly at the TIR areaunder observation. Again by naked eye, adjust its lateral position withtranslators on the focusing lens mount (not with the mirrors control-ling the raw laser beam) so that the TIR region occurs directly in linewith the objective. To guide this adjustment, look for three closelyaligned spots of scattered light, corresponding to where the focusedbeam first crosses the immersion oil layer, where it totally reflects offthe sample surface, and where it exits by recrossing the oil.

8. The TIR region should now be positioned well enough to appear inview in the microscope when viewed as fluorescence with the stan-dard filters in place. In general, the TIR region will appear as a yellowellipse or streak. Make final adjustments with the focusing lens to centrethis area. The TIR area can be distinguished from two out-of-focusblurs past either end of the ellipse or streak (arising from autofluor-escence of the immersion oil) because the TIR spot contains sharply

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focused images of defects in the Dil coating. The focusing lens can bemoved forward or backward along the laser optical path to achieve thedesired size of the TIR area.

9. With the optics now correctly aligned for TIR, translate the prismvertically to remove the Dil sample, and replace it with the actual cellsample. Relower the prism to make optical contact. Although the TIRregion will not be exactly in the same spot (because of irreproducibil-ity in the prism height), it will be close enough to make final adjust-ments with the focusing lens while observing fluorescence from thecell sample.

3.4 Inverted microscope TIR without a prismBy using an objective with a numerical aperture of 1.4 (the highest commer-cially available), supercritical angle incident light can be cast upon the sampleby epi-illumination through the objective. The incident beam must be con-strained to pass through the periphery of the objective's pupil and mustemerge with only a narrow spread of angles; this can be accomplished byassuring that the incident beam is focused off-axis at the objective's back focalplane. It emerges into the immersion oil (n3 = 1.52) at a maximum angle 0given by:

For total internal reflection to take place at the sample surface, 6 must begreater than the critical angle 0C given by:

From Equations 6 and 7, it is evident that the NA must be greater thann1, preferably by a substantial margin. This is no problem for an interfacewith water with n1 - 1.33 and a NA = 1.4 objective. But for viewing the in-side of a cell at n1 — 1.38, this configuration is marginal at best for producingTIR.

The advantages of prismless inverted TIR include the possibility of viewinga sample that is completely accessible from the top, and the compatibility (infact necessity) for using the highest aperture, highest resolution, brightestobjectives available. The arrangement is also easily compatible with intersect-ing beams to produce interference fringes: the high mechanical stabilityenables one to achieve interfringe spacings of ~ 0.3 (um without the blurringeffects of small vibrations. Three possible arrangements for prismless TIR areshown in Figure 8: one as described in the Protocol 2; one for producing inter-ference fringes at the TIR surface; and one which utilizes a conventional arcsource rather than a laser beam.

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Figure 8. Three TIR configurations G, H, and I for an inverted microscope and a NA = 1.4aperture objective but no prism. The microscope epi-illuminator system is drawnschematically here and connected with configuration H, but the same system is impliedfor the extensions of configurations G and I. In configuration G, two beams (split from thesame laser) intersect at the field diaphragm, forming an interference pattern there and onthe sample. In configuration I, an arc lamp rather than a laser is used. An opaque diskmust be used here to block subcritical arc lamp light. The conical prism serves only toincrease the light flux at supercritical angles so that less energy is lost at the opaque disk.

Protocol 2. Alignment of prismless TIR

Equipment• Laser with blue or green output, at least 1

W total power• Inverted fluorescence microscope equipped

with epi-illumination dichroic mirror andbarrier filter appropriate for the lasercolour, but with the excitation filter re-moved

• Objective with numerical aperture of 1.4

• Assorted optical mounts• Plano-convex lens of short radius of curva-

ture or, alternatively, a hemispherical or tri-angular prism

• Converging lens of several centimetresfocal length

• Safety goggles

Method

1. Place a bare coverslip on the microscope stage (with immersion oilbetween the objective and the coverslip), and focus on its uppersurface.

2. Remove all obstructions between the coverslip sample and the ceiling.Allow a collimated laser beam (the 'raw' beam) to enter the standard

D. Axelrod


epi-illumination port field diaphragm along the optical axis. A largearea of laser illumination will be seen on the ceiling, roughly straightup.

3. Place the triangular or hemispherical prism or plano-convex lens (flatside down) on the coverslip over the objective, making optical contactwith a layer of immersion oil. This prism or lens is not going to beused in actual experiments. It is used here to avert total internalreflection and thereby couple light out of the coverslip and onto thewall or ceiling of the room. Here again, safety goggles are advisable.

4. Reposition the laser beam with mirrors positioned upbeam from themicroscope so that the beam still enters the centre of the field dia-phragm but now at a small angle to the optical axis. This angle shouldbe continuously adjustable; slowly increase the angle. The ceilinglaser illumination will 'set' to an ever-lower position on the wall untilit just disappears. At this angle, it is just blocked by the internal aper-ture of the objective. Back off the entrance angle so that half theilluminated area is seen.

5. Place the converging lens about 20 cm 'upbeam' from the fielddiaphragm and concentric with the incoming beam. The illuminatedregion on the wall will now be a different size, probably smaller.

6. Move the converging lens longitudinally (along the axis of the laserbeam) to minimize the illuminated region on the wall. This will occurwhere the converging lens focal point falls exactly at a plane outsidethe microscope equivalent to the objective's back focal plane. At thisposition, the beam is thereby also focused at the objective's actualback focal plane and emerges from the objective in a roughly colli-mated form.

7. Fine-tune the lateral position of the converging lens and the rawbeam mirrors so that the beam on the wall is just barely above thepoint at which it disappears. This ensures that the beam is propagat-ing up along the inside periphery of the objective.

8. Verify that TIR is achievable by moving to the sample to a clean newspot not under the prism. No light should emerge (except for somescattering) because of total internal reflection at the glass coverslip/air interface.

9. Replace the bare coverslip with an identical one coated with Dil (seeProtocol 7). Place a droplet of water on the Dil coverslip directlyabove the objective, and again verify that no light emerges even atthis glass/water interface.

10. View the Dil fluorescence through the microscope; the illuminatedregion should be circular or elliptical and roughly centred. Fine-tune

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the mirrors and converging lens to centre the TIR fluorescence in thefield of view.

11. The size of the TIR fluorescence area on the sample is directly propor-tional to the laser beam size at the field diaphragm. To change this size,replace the converging lens with another one of different focal length,but always keep its focal point at the objective's equivalent back focalplane, which is at a fixed position upbeam from the microscope.

12. Replace the Dil coverslip with the actual cell sample. When the cellsare in-focus, the TIR optics should be perfectly aligned without needof further adjustment.

13. If switching back and forth between TIR and epi-illumination is de-sirable, design the optics with movable mirrors (within easy reach)that can select angular or straight-on laser paths.

14. If interference fringe TIR is desirable, use a beam splitter and arrangethe second beam to enter the centre of the field diaphragm from anapproach at the same angle but at different azimuthal position aroundthe optical axis. A relative azimuthal angle of 180° will give the closestspaced fringes. It may be most convenient to use the same converginglens for both beams. Then the converging lens should be positionedon-axis but each beam should enter it off-axis so that each still arrivesat the centre of the field diaphragm. Be sure that any difference inpath length of the two beams from the beam splitter to their re-intersection point is less than the coherence length of the laser (a fewmillimetres or centimetres); otherwise, no interference will occur.

3.5 Rapid chopping between TIR and epi-illuminationRegardless of the method chosen to produce TIR in a microscope, it is oftendesirable to switch rapidly between illumination of the surface (by TIR) anddeeper illumination of the bulk (by standard epifluorescence). For example, atransient process may involve simultaneous but somewhat different fluor-escence changes in both the submembrane and the cytoplasm, and both mustbe followed on the same cell in response to some stimulus (3). Figure 9 showsa method using computer driven acousto-optic modulators by which the rapidchopping can be done.

4. General experimental suggestionsRegardless of the optical configuration chosen, the following suggestions maybe helpful.

(a) The prism used to couple the light into the system and the (usually dis-posable) slide or coverslip in which TIR takes place need not be matchedexactly in refractive index.

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Figure 9. Optoelectronic system used for rapid chopping between TIR and EPI. The zeroorder diffraction direction of acousto-optic modulator 1 (AOM1) is the TIR path; the firstorder is the EPI path. AOM 2 and AOM3 serve only to increase the on/off contrast of theEPI and TIR beams, respectively, so that they cleanly alternate. The computer that con-trols the square wave pulses to the AOMs also acquires data from the single-channelphoton counter so that the time course of the two illumination modes can be separated insoftware.

(b) The prism and slide may be optically coupled with glycerol, cyclohexanol,or microscope immersion oil, among other liquids. Immersion oil has ahigher refractive index (thereby avoiding possible TIR at the prism/coupling liquid interface at low incidence angles) but it tends to be moreautofluorescent (even the 'extremely low' fluorescence types).

(c) The prism and slide can both be made of ordinary optical glass for manyapplications, unless shorter penetration depths arising from higher refract-ive indices are desired. Optical glass does not transmit light below about310 nm and also has a dim autoluminescence with a long (several hundredmicrosecond) decay time, which can be a problem in some photobleach-ing (FRAP) experiments (see Section 5d). The autoluminescence of highquality fused silica (often called 'quartz') is much lower. Tissue culturedish plastic (particularly convenient as a substrate in the upright micro-scope set-up) is also suitable, but tends to have a significant autofluor-escence compared to ordinary glass. Different brands of tissue cultureplastic have significantly different amounts of autofluorescence; Corningbrand is one of the least fluorescent. More exotic high n3 materials such as

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sapphire, titanium dioxide, and strontium titanate can yield exponentialdecay depths d as low as l0/20.

(d) The TIR surface need not be specially polished: the smoothness of astandard commercial microscope slide is adequate.

(e) Illumination of surface adsorbed proteins can lead to apparent photo-chemically-induced cross-linking. This effect is observed as a slow, continual,illumination-dependent increase in the observed fluorescence. It can beinhibited by deoxygenation (aided by the use of an O2-consuming enzyme/substrate system such as protocatachuic deoxygenase/protocatachuicacid or a glucose/glucose oxidase system), or by 0.05 M cysteamine.

(f) Virtually any laser with a total visible output in the 0.5 W or greater rangeshould be adequate. The most popular laser for cell biological work with amicroscope appears to be a 3 W continuous wave argon laser.

(g) TIRF experiments often involve specially coated substrates. A glass sur-face can be chemically derivatized to yield special physi- or chemi-absorp-tive properties. Covalent attachment of certain specific chemicals areparticularly useful in cell biology and biophysics, including: poly-L-lysinefor enhanced adherence of cells; hydrocarbon chains for hydrophobicizingthe surface in preparation for lipid monolayer adsorption; and antibodies,antigens, or lectins for producing specific reactivities. Derivatizationgenerally involves pre-treatment of the glass by an organosilane.A planar phospholipid coating (possibly with incorporated proteins) onglass can be used as a model of a biological membrane. Methods forpreparing such model membranes on planar surfaces suitable for TIR arereviewed in ref. 4.Aluminium coating (for surface fluorescence quenching; see Section 2.3)can be accomplished in a standard vacuum evaporator; the amount ofdeposition can be made reproducible by completely evaporating a pre-measured constant amount of aluminium. After deposition, the uppersurface of the aluminium film spontaneously oxidizes in air very rapidly.This aluminium oxide layer appears to have some similar chemical prop-erties to the silicon dioxide of a glass surface; it can be derivatized byorganosilanes in much the same manner.

(h) The TIRF spot should be focused to a width no larger than the field ofview; the larger the spot, the more that spurious scattering and out-of-focus fluorescence from the immersion oil layer between the prism andcoverslip will increase the generally very low fluorescence backgroundattainable by TIRF.

(i) A laser source is generally preferable to an arc lamp for TIRF, becausecollimation of arc lamp light entails a great loss of intensity. On the otherhand, laser illumination suffers from unavoidable interference fringing onthe sample. This can be minimized by very clean optics. But for critical

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D. Axelrod

applications, it may be advisable to perform rapid jiggling of the beamor to compute a normalization of sample digital images with the controldigital image of a uniform concentration of fluorophores.

(j) The incidence angle should exceed the critical angle by at least a coupleof degrees. At incidence angles very near the critical angle, the cells cast anoticeable 'shadow' along the surface.

5, Applications of TIRF microscopyApplications of TIRF microscopy in cell biology include:

(a) Localization of cell/substrate contact regions in cell culture. Quantitativedetermination of the absolute distance from the surface to a labelled cellmembrane at a cell/substrate contact region can be based on the variationof the emitted fluorescence with 0. This effort is challenging because cor-rections have to be made for 6-dependent reflection and transmissionthrough four stratified layers (glass, culture medium, membrane, andcytoplasm), all with different refractive indices. A variation of TIRF toidentify cell/substrate contacts produces essentially a negative of thestandard fluorescence view of labelled cells (5). The solution surroundingthe cells is doped with a non-adsorbing and non-permeable fluorescentvolume marker, fluorescein labelled dextran. Focal contacts then appearas dark areas and other areas appear brighter, depending on the depth ofsolution illuminated by the evanescent wave in the cell/substrate gap.

(b) High contrast visualization of submembrane cytoskeletal structure onthick cells. Optical sectioning by TIRF is particularly useful in viewingsubmembrane cytoplasmic filaments on thick cells. Epi-illuminationexcites fluorescence from out-of-focus planes and leads to a diffuse fluor-escence that obscures detail. Although TIRF cannot view deeply into thecell, it can display the submembrane filament structure with high contrast.

(c) Time lapse fluorescence movies. Epifluorescence illumination appears toadversely affect cell viability, even with occasional exposure for makingmovies. TIRF seems particularly advantageous for long-term viewing ofcells, since the evanescent wave minimizes exposure of the cells'organdies to excitation light.

(d) Desorption kinetic rates of reversibly bound biomolecules at biologicalmembrane surfaces. If the evanescent wave intensity is briefly flashedbrightly, then some of the fluorophores associated with the surface will bephotobleached. Subsequent exchange with unbleached dissolved fluoro-phores in equilibrium with the surface will lead to a recovery of fluores-cence, excited by a continuous but much attenuated evanescent wave.The time course of this recovery is a measure of the desorption kineticrate (6). (This technique is called TIR/FRAP in reference to fluorescence

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recovery after photobleaching.) Kinetic rates of hormones and cytoskele-tal proteins at model membranes, flattened biological membranes, andintact cell surfaces have been measured by this technique. Kinetics at thecytofacial surface of intact cells can be viewed employing TIR/FRAP oncells that have been microinjected with a labelled molecule of interest.

(e) Surface diffusion of reversibly bound biomolecules at biological membranesurfaces. TIR/FRAP can be used to measure surface diffusion coefficientsalong with on/off kinetics, if the evanescent wave intensity is variegatedover a distance on the surface short compared to the characteristic dis-tance covered by surface diffusion within the time available before des-orption. One way of producing this variegation is simply by focusing;another way is by interfering two TIRF beams, as discussed with con-figuration D (Section 3.1.4); a third way is by placing a striped patternRonchi ruling in the incident beam at a position where it forms a realimage on the sample.

(f) Orientational distributions of fluorescent molecules at a surface. Thepolarization properties of the evanescent wave can be used to exciteselected orientations of fluorophores. Standard polarized epi-illuminationcannot distinguish order from disorder in the polar direction (defined asthe optical axis) because epi-illumination is polarized transverse to theoptical axis. But microscope TIR illumination can be partially polarizedin the optical axis direction (the z direction) and can thereby detect orderin the polar direction. Note that fluorescence polarization detectedthrough a microscope should be corrected for the slight depolarizingeffect of high aperture objectives (7).

(g) Reduction of cell autofluorescence relative to fluorescence excited atcell/substrate contacts.

(h) Viewing a cell bound fluorescent ligand in the presence of the same ligandin the bulk. Many cell surface receptors bind their ligands reversibly, so asurface bound population can be maintained only with a substantial con-centration in the bulk. In some other cases, the cell surface receptor bindsligands irreversibly but then the complex is internalized and replaced by afresh unbound receptor. The newly appearing receptors can be labelledcontinuously with ligand in the bulk. The bulk fluorescence will be dis-criminated against by TIRF.

(i) Observing rapid dynamical processes at biological membranes. TIRF hasbeen used to quantitatively compare intracellular ionic concentrationtransients proximal to the membrane versus deeper in the interior (3). Ithas also been used qualitatively to observe fluorescence loaded secretoryvesicle fusion with the membrane (8).

(j) Reduction of instrumental luminescence background. In standard epi-illumination, the full power of the illumination light excites some fluores-cence from the glass and glues in the objective. In TIR, this background is

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D. Axelrod

eliminated, thereby making fluorescence detection of single moleculesmore feasible (9, 10). Related to this application is the capability of seeingfluorescence fluctuations as fluorescent molecules enter and leave the thinevanescent field region in the bulk. These visually obvious fluctuationscan be quantitatively autocorrelated to obtain kinetic information aboutthe molecular motion ('fluorescence correlation spectroscopy', FCS).

(j) Fluorescence lifetimes at surfaces. TIR has been combined with fluor-escence lifetime imaging of cultured cells labelled with a terbium chelate(11). Such chelates have a very long (millisecond) lifetime compared withorganic fluorophores, but as techniques for measuring fast fluorescencelifetimes in a microscopic spatially resolved region are further developed,they will be readily adapted to TIRF.

6. Comparison with other optical sectioningmicroscopies

Confocal microscopy (CM) is another technique for apparent optical section-ing, achieved by exclusion of out-of-focus emitted light with a set of imageplane pin-holes. CM has the clear advantage in versatility; its method of opti-cal sectioning works at any plane of the sample, not just at an interfacebetween dissimilar refractive indices. However, other differences exist which,in some special applications, can favour the use of TIRF:

(a) The depth of the optical section in TIRF is ~ 0.1 um whereas in CM it is arelatively thick ~ 0.6 um.

(b) In some applications (e.g. FRAP, FCS, or on cells whose viability isdamaged by light), illumination and not just detected emission is bestrestricted to a thin section; this is possible only with TIRF.

(c) Since TIRF can be adapted to and made interchangeable with existingstandard microscope optics, even with 'home-made' components, it ismuch less expensive than CM. However, at the time of this writing, TIRFmicroscopy modules or kits are not commercially available; the mirrorsand prisms must be purchased separately from optical supply companiesand configured by the end user.

Cell/substrate contacts can be located by a non-fluorescence techniquecompletely distinct from TIRF, known as 'interference reflection microscopy'or 'reflection contrast microscopy' (RCM). Using conventional illuminationsources, RCM visualizes cell/substrate contacts as dark regions. RCM has theadvantage that it doesn't require the cells to be labelled, but the disadvan-tages that it contains no information of biochemical specificities in the contactregions and that it is less sensitive to changes in contact distance (relative toTIRF) within the critical first 100 nm of the surface.

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AcknowledgementsThe author is grateful to Susan Sund and Rhonda Dzakpasu for useful discus-sions. Figure 1 was provided by Susan Sund. This work was supported by agrant NSF MCB9405928.

References1. Axelrod, D., Hellen, E.H., and Fulbright, R.M. (1992). In Topics in fluorescence

spectroscopy, Vol. 3: biochemical applications (ed. J.R. Lakowicz), p. 289. PlenumPress, New York.

2. Burghardt, T.P. and Thompson, N.L. (1984). Opt. Eng., 23, 62.3. Omann, G.M. and Axelrod, D. (1996). Biophys. J., 71, 2885.4. Thompson, N.L., Pearce, K.H., and Hsieh, H.V. (1993). Eur. Biophys. J., 22, 367.5. Gingell, D., Heavens, O.S., and Mellor, J.S. (1987). J. Cell Sci., 87, 677.6. Thompson, N.L., Burghardt, T.P., and Axelrod, D. (1981). Biophys. J., 33, 435.7. Axelrod, D. (1989). In Fluorescence microscopy of living cells in culture. Part B

(ed. D.L. Taylor and Y.-L. Wang), Meth. Cell Biol. 30, p. 333. Academic Press,New York.

8. Steyer, J.A., Horstmann, H., and Aimers, W. (1996). Nature, 388, 474.9. Vale, R.D., Funatsu, T., Pierce, D.W., Romberg, L., Harada, Y., and Yanagida, T.

(1996). Nature, 380, 451.10. Dickson, R.M., Norris, D.J., Tzeng, Y.-L., and Moerner, W.E. (1996). Science, 274,

966.11. Phimphivong, S., Kolchens, S., Edmiston, P.L., and Saavedra, S.S. (1995). Anal.

Chim. Acta, 307, 403.

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Nanovid microscopyGRETA M. LEE

1. IntroductionNanovid microscopy, nanometre-sized particles viewed with video-enhancedmicroscopy (1-3) is used to directly observe the lateral mobility of goldlabelled membrane proteins and lipids, to observe the precise location andmobility of gold labelled pericellular and extracellular matrix macromolecules,and to follow endocytosis of gold labelled molecules. The technique involvesthe use of 30-40 nm colloidal gold particles conjugated to antibodies, strepta-vidin, lectins, or other ligands. The association of the antibody to the gold isnon-covalent but extremely stable. Since a single colloidal gold particle is typi-cally conjugated to many antibody molecules (4), an individual gold particlemay bind to a small group of like membrane molecules rather than to a singlemolecule (5). The gold tag is visualized using video contrast enhancement todetect the light scattering of the gold particle. The gold can be viewed withbright-field, DIC, or epipolarization optics. Examples of bright-field and DICimages are shown in Figure 1.

Individual 20-40 nm gold particles are not directly visible by eye throughthe oculars but can be detected with video contrast enhancement of the Airydisk (see Chapter 3) produced by light scattering of the gold particle. Videocontrast enhancement involves both camera (analogue) and image processor(digital) adjustments as described below. The degree of scattering is propor-tional to the sixth power of the particle radius and to the fourth power of theilluminating wave number (the reciprocal of the wavelength) (2). Because thelight scatter is so strong, gold particles appear much larger in the video imagethan in electron micrographs that show the true size relative to the cell. This isdemonstrated in Figure 2 where the same cell is viewed by video light micro-scopy and electron microscopy (prepared as described in ref. 6). Note howmuch smaller the gold particles are relative to the cell in the electron micro-graph. Also note that similar sized gold particles can have varying levels ofcontrast in the video image. This can be due to slight variations in focal level.In addition, single gold particles cannot be distinguished from doublets in thevideo image, but three adjacent gold particles have higher contrast and appearas a larger spot. Whether two or three gold particles are imaged as a single

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Greta M. Lee

Figure 1. Examples of colloidal gold labelling of the plasma membrane (A, B) and thepericellular matrix (C, D). (A) and IS) show a lamella of a C3H-10T1/2 fibroblast labelledwith 30 nm colloidal gold conjugated to an antibody to Thy-1, a glycophosphatidylinositol-linked protein found on the cell surface. Note how the gold particles are easilyrecognized on the cell surface in the bright-field image (A) but are difficult to distinguishfrom intraceflular structures in the DIC image (B). The gold particles do not align in thetwo images because they were mobile. (C) and (D) show a bovine chondrocyte labelledwith 40 nm colloidal gold conjugated to an antibody to type VI collagen. The label isfound in the pericellular matrix which is a three-dimensional structure extending outfrom this rounded cell. Individual gold particles are readily detectable in the bright-fieldimage (C) but are difficult to to see in the DIC image (B). Arrowheads indicate cell edges.All figures are at the same magnification. Bar = 2 um.

spot or as separate spots is dependent on their spacing and on the resolutionlimit of the optics. For two adjacent points to be resolved as separate points,the centres of the Airy disks for those two points must be separated by a mini -mum distance determined by the resolving power of the optics (see Chapter 1),Thus an objective and a condenser with high numerical aperture will be hetlerfor resolving closely spaced gold particles.

The variation in image size for single gold particles and the inability to dis-tinguish between single and two closely spaced gold particles make doublelabelling with colloidal gold of two different sizes impractical with video-enhanced light microscopy. However, colloidal gold can be used with afluorescent probe or 1-2 um latex beads.

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12: Nanovid microscopy

Figure 2. Bright-field video image of a region of a gold labelled cell before (A) and afterimage processing for automatic detection of gold particles (B), The electron micrograph(C) of the boxed region in (B) demonstrates that individual 30 nm gold particles can bedetected with a video microscope system. The lower case letters indicate correspondingsingle gold particles (a-d) and aggregates (e and f). This C3H fibroblast was labelled with30 nm gold anti-pgp-1, which binds to an integral membrane glycoprotein. After fixationand imaging by nanovid microscopy, the cell was processed for whole mount trans-mission electron microscopy. The distances between the gold particles are shorter in theelectron micrograph than in the video images due to cell shrinkage during the dehydra-tion steps. Bars (B) = 2 um; (C) = 0.2 um. Reproduced from ref. 9 with permission.

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1.1 Use of nanovid microscopy to analyse molecularmobility in membranes

To analyse the pattern of movement of the labelled molecule, a series ofimages over time is recorded. The time frame for recording depends on therate of movement of the labelled molecules and on the question being asked.For fast diffusing molecules, such as lipids in a membrane, images are re-corded at video rate. For slower moving molecules, such as capping of mem-brane proteins, images may be recorded at one frame per second or slower.The sophistication of the analysis can range from tracing the movement ona video monitor with a felt tip marker to analysing the centroids (x, y co-ordinates of the centre) of each gold particle in each image. For the latter, theanalysis can involve:

(a) Manually locating the centroid with a pointer.

(b) Manually identifying the particles to be tracked and using an image pro-cessing program to compute the centroid.

(c) Using the image processor to both identify the particle and compute thecentroid.

The centroids are then used to compute the trajectory of the labelledmolecule. These trajectories are used to analyse the pattern of movementand/or to compute the diffusion coefficient. One of the purposes of thisapproach is to directly observe intramembrane domains or 'corrals' as well asfree diffusion and directed transport within the plane of the membrane.Unfortunately, Brownian motion produces trajectories with a variety of ran-dom patterns (7, 8) that can complicate the detection of larger domains (9).Thus the interpretation of the data requires careful statistical analysis (5, 10,11). With more sophisticated analysis, temporary confinement can be sepa-rated from pure random motion (12, 13).

1.2 Use of nanovid microscopy to study the extracellularmatrix

Another, less widely recognized, use of nanovid microscopy is to study loca-tion, mobility, and assembly of extracellular matrix molecules on living and fixedcells. This approach is especially useful for studying the pericellular matrix onliving cells because this matrix is made up of hydrated hyaluronan and proteo-glycans which collapse with fixation and dehydration. Nanovid microscopywas used to analyse the roles of hyaluronan and aggrecan in forming thethree-dimensional, dynamic pericellular matrix on chondrocytes (14). Col-loidal gold particles can also be used as markers on extracellular matrix fibrils,such as collagen fibrils, to follow their movements on the cell surface as thecell arranges its extracellular matrix (Lee and Loeser, submitted).

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Protocol 1. Probe preparation: method for conjugating purifiedantibodies to 30 or 40 nm colloidal golda

Equipment and reagents96-multiwell platepH meterMicrocentrifugePurified monoclonal or polyclonal anti-body, dialysis tubing, and clips30 or 40 nm colloidal gold20M Carbowax

• 10% bovine serum albumin (BSA) dis-solved in dH2O

• Glycerol• 2 mM borate buffer pH 8.9. 0.2 M K2C03, 0.2 M H3PO4

. 20 mM Tris, 150 mM NaCI pH 8.2

Method

1. Dialyse the antibody (roughly 300 ul of 0.1 mg/ml) against 2 mMborate buffer pH 8.9, for 1 h at 4°C. Centrifuge the protein to removeany aggregates.

2. Adjust 10 ml colloidal gold suspension to pH 8.9 with 0.2 M K2CO3 and0.2 M H3P04. Caution: gold plugs non-gel type pH electrodes. Workquickly and soak the electrode in pH 4 citrate buffer immediately afteruse. Alternatively, use pH paper.

3. Do a protein concentration curve: put a 96-multiwell plate on a whitesurface. Using five to ten wells, do a dilution series of the proteinadding 10 ul to each well. Add 100 ul colloidal gold to each. Mix well.Wait 10 min. Add 10 ul 10% NaCI, mix, wait 5 min. Look at the colour—pink indicates enough protein to stabilize 100 ul of gold, blue indicatesaggregation of the gold due to an inadequate amount of protein.Select the lowest concentration of protein which gives a pink colour asthe concentration to use.

4. Calculate the volume of protein necessary to stabilize the remaininggold solution or the volume of gold that can be stabilized with theamount of protein available. Add the dialysed protein to the goldsuspension, vortex for 2 min, let sit at room temperature for 8 min.

5. Stabilize the gold by adding 10% BSA to 1%, and 1% 20M Carbowax to0.05%. Centrifuge at 15000 g for 12 min. Discard the supernatant andresuspend the pellet in 0.05% Carbowax, 1% BSA, 20 mM Tris, 150mM NaCI pH 8.2. Give two more washes in the same solution toremove any unbound antibody.

6. Resuspend the pellet in a small quantity (100-500 ul) of the abovesolution (the gold suspension should be the colour of port wine). Addglycerol to 50%. Store at-20°C.

• Adapted from Aurobeads colloidal gold for macromolecule labelling, by Janssen.

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General tips:

(a) Filter all solutions with a 0.22 um filter. Particulates can cause the gold toaggregate and bacteria will be concentrated with the gold by the washsteps.

(b) As an alternative to using incredibly clean glassware, use sterile, disposableplastic test-tubes.

(c) To reduce the sticking of conjugated gold to the sides of Eppendorf tubesduring centrifugation, add the wash solution first.

(d) Osmolarity is important for antibody stability. Before and after con-jugation to gold, minimize the time antibodies are left in very dilute saltsolutions. An antibody that has lost its proper conformation by prolongedexposure to dilute solution will have little to no antigen affinity afterconjugation to the gold.

(e) To prepare paucivalent gold probes (5), mix an irrelevant antibody withthe desired antibody before adding to the gold. Do a protein concentra-tion curve on each to determine the correct ratio for mixing.

Protocol 2. Cell culture and labelling

Equipment and reagents• Coverslips and glass scribe to cut spacers « Tissue culture mediumb• Silicon grease or Parafilm • 10% BSA• 30-40 nm colloidal gold conjugated to a pri- • VALAP: vasoline:lanolin:paraffin (1:1:2, by

mary antibody, streptavidin,a or a lectin weight)

Method

1. Grow cells in a monolayer on No. 1 22 mm2 coverslips. (Ideally, a No.1.5 coverslip should be used, but in this thickness category there are afew coverslips that are too thick for focusing with a high NA oilimmersion objective. Use of No. 1 coverslips avoids the frustration.)

2. Remove the glycerol and any unbound antibody by adding 5-20 ulof gold suspension (as prepared in Protocol 7) for each coverslip to500 ul tissue culture medium containing 1% BSA. Centrifuge as inProtocol 1. Repeat the wash step once or twice.

3. Resuspend the pellet in the desired volume, approx. 100 ul per cover-slip.

4. Place two No. 1 or No. 2 coverslip strips as spacers on a clean glassslide; a thin film of silicon vacuum grease can be used to hold them inplace. Alternatively, two strips of Parafilm can be used as spacers.

5. Place 100 ul of gold suspension on the slide between the spacers, andinvert the coverslip with cells onto the drop.

430


6. Incubate for 20 min or longer in a moist chamber at room tempera-ture or 37°C depending on your experiment.

7. Carefully dry the sides with the spacers then seal with melted VALAP.

8. Slowly flow 200 ul of tissue culture medium with BSA or serumthrough the chamber to rinse out unbound gold. Flowing mediumthrough too quickly can shear the cells.

9. Dry the remaining edges of the coverslip and seal with VALAP. Avacuum flask attached to the house vacuum line and with a Pasteurpipette inserted in the intake tubing works well for drying.

10. Clean the top of the coverslip with a small drop of dH20.

11. Prepare a control coverslip without colloidal gold for comparisonpurposes.

* When using streptavidin gold, the cells need to first be labelled with a biotinylated probe.b Hepes-buffered Ham's F-12 (Sigma, N8641) is a good medium as the the bicarbonate con-centration is low enough that fair pH control can be maintained outside of a CO2 atmosphere.

Protocol 3. Viewing the labelled cells

Equipment• Microscope with DIC optics, high NA, oil im-

mersion condenser and objective, a greenband pass filter, and IR (e.g. Schott KG5)and UV (e.g. Schott GG475) filters, 2-4 xadapter for the video camera, and stabilizedlight source

• Video camera with manual gain and offsetadjustments

• Video monitor

Computer with frame grabberImage processor software capable of handl-ing real time background subtractionImage storage device (e.g. VCR, large harddisk, Zip drive, optical disk recorder)Air curtain incubator or heated stage andobjective for live mammalian cells

Method

1. Place the specimen on the stage. For an inverted microscope, place adrop of oil on the slide and bring the condenser lens into contact withthe oil. For an upright microscope, place a drop of oil on the con-denser and bring the oil into contact with the slide.

2. With x 20 objective and phase-contrast or DIC optics, focus on thespecimen. (See Chapter 1, Protocol 9 for instructions in using DICoptics.)

3. Adjust the condenser for Kohler illumination (Chapter 1, Protocol 2).

4. Locate a cell of desired morphology.

5. Switch to the x 100 objective using a small drop of oil on theobjective and focus on the specimen with DIC optics.

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Greta M. Lee


6. Readjust the condenser for Kohler illumination.

7. Obtain a good image of the desired cell on the video monitor. Savethe image to disk for future reference.

8. Reduce the light intensity and then go to bright-field optics by re-moving the analyser from the light path.

9. If the camera has an indicator for too much light, or if you have anoscilloscope, or your software has a live histogram function, increasethe light intensity to the maximum allowable for the camera or ashigh as possible without saturating the pixels.

10. Use analogue contrast enhancement to view the gold on the videomonitor. Adjust the camera by increasing the gain to give a brightimage then bringing up the black level until the screen appears anormal grey. (Do not overly increase the gain as the resulting in-crease in noise will make analysis more difficult.) If the gold particlesare not visible, the adjustments are repeated until any dirt specks onthe camera face plate are displayed on the monitor.

11. Delicately adjust the fine focus until gold particles are visible as smallblack spots.a Small organelles within the cell may also show up. Withbright-field, the organelles can be distinguished from the gold byslight focal adjustments which make the gold particle disappear butchange the organelles from black to white (1).

12. Use digital contrast enhancement to further improve the image. Abackground image (a blank area only slightly out-of-focus) is sub-tracted, and then the contrast is enhanced by adjusting the gain andoffset of the image with the image processor.

• If the selected cell is not labelled, there are usually gold particles that are non-specificallyattached to the substratum that can be imaged. These also serve as a reference for distinguish-ing gold particles from organelles. Examine the control slide without gold labelling to improveconfidence in distinguishing gold from organelles.

2. General information on equipment and methods2.1 MicroscopeBoth bright-field and DIC microscopy can be used. With bright-field, more ofthe gold particles show up and the gold is easier to distinguish from organ-elles; however, the optical sectioning is not quite as good as with DIC. Whenusing bright-field to view the gold, it is useful to record corresponding DICimages to provide information regarding location on the cell, cell shape, etc.For bright-field, having the polarizer and Wollaston prisms in the light pathdoes not seem to affect the image quality.

432


All lenses and filters in the light path need to be clean because the contrastenhancement necessary to see the gold particles will also show up any dirt.The use of a 2-4 X adapter or projection lens for the camera is dependent onthe size of the imaging area of the camera and whether individual gold par-ticles are to be tracked or just visualized. With a 1" newvicon camera, theadapter is needed for tracking but is not necessary for localization. A 1/3"CCD chip gives a higher magnification in the video image and thus theadapter may not be needed. The light source needs to be extremely stable ifimage analysis for tracking of bright-field images is to be semi-automated.This is because slight variations in grey level due to light fluctuations areamplified by the processing needed for the image processor to detect the goldparticles. Thus the light fluctuations result in images that are too dark or lightfor semi-automated analysis.

2.2 CameraA video camera is used to both visualize the gold and to give analogue con-trast enhancement. A newvicon tube camera with manual adjustments of gainand black level works well, but there is variation in how far the gain and blacklevel can be adjusted among different manufacturers. Thus, it is wise to testthe camera before purchase. A method to indicate adequate and excess lightto the camera is extremely helpful for bright-field adjustments. For imageacquisition of rapidly moving particles, we have found the Hamamatsu C2400performs well. A CCD camera with both gain and offset or black level adjust-ments can also be used. Some CCD cameras have a mottle on the face platethat is visible at high contrast. Output rate of some of the high resolution digitalCCD cameras may be a limiting factor for nanovid microscopy because pre-cise focusing is difficult at less than 15 frames per second. There is adequatelight especially with bright-field microscopy, and thus cooled, integratingcameras designed for low light level work are not needed. On the other hand,sensitivity to electronic noise is important in this application because the con-trast is pushed so high that all noise is also amplified making subsequentimage analysis difficult. The presence of electronic noise also appears to be afunction of the electrical grounding within the microscope as we have foundthat cameras attached to an Olympus 1X70 microscope pick up very littleelectronic noise.

2.3 Image processor and frame grabberFor acquiring images of particles diffusing at 1-10 X 10~9 cm2/sec, real timebackground subtraction and digital contrast enhancement are needed. Realtime image averaging capabilities are helpful for slower moving gold particles.This can be typically performed faster on the frame grabber board than inthe computer. We have found that Image-1 from Universal Imaging (WestChester, PA) simultaneously performs real time background subtraction,

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Greta M. Lee

contrast enhancement, and image storage to an optical disk recorder. Meta-Morph, also from Universal Imaging, is supplied with different video boardsand thus real time background subtraction and enhancement may not beavailable. NIH-Image, freeware available for both the Macintosh and the PCcan also be used for image acquisition and analysis. The Scion AG-5 board(Macintosh) is compatible with NIH-Image and newvicon and video CCDcameras and allows video rate background subtraction and frame averaging,but there is only digital output of the processed image which means theimages must be stored on a computer prior to making a video tape.

2.4 Image storageDepending on the recording rate, images can be stored on a hard disk drive,an optical memory disk recorder (Panasonic), or video tape. An optical diskrecorder gives video rate storage and retrieval, the resolution is good, andimages can be retrieved individually and selected by the image processor;however, the initial cost is high. For movies, when later analysis is not desired,half-inch VHS is acceptable, but S-VHS is better if the recording will becopied later. With NIH-Image and the Scion AG-5 board, cropped images canbe stored to the hard disk at or near video rate. Video rate storage is onlyessential for fast diffusing particles (i.e. gold labelled lipids in a membrane).For slower moving particles, a running average of two frames will give asharper image, and images can be saved at ten or less frames per second. Aminimum of 200 images in a series is recommended for analysing trajectoriesof individual gold particles. An Iomega Jaz drive with 1 GB disk cartridges isa simple way to store large numbers of digitized images. Alternatively, awritable CD can be used as an economical storage medium.

3. Image analysis to analyse molecular mobility(single particle tracking)

To track the movement of individual gold particles, the centroids of each goldparticle of interest needs to be located in a series of images. One can manuallypoint to each particle with a cursor to find the centroids, but this is a tediousand difficult process. The alternative is to use the image processor to auto-matically locate the particles with user-defined selection criteria. For bright-field images, the gold particles need to be separated from the background bythresholding. A histogram function for adjusting grey levels can also be help-ful. Smoothing or low pass filters make the gold particles appear more uni-form. For the actual determination of the centroids with Image-1's 'objectmeasurement mode' or NIH-Image's 'particle analysis', parameters must firstbe set to define size and shape limits so that the gold particles can be distin-guished from noise and to eliminate aggregates. DIC images do not haveenough contrast for use with the object measurement mode or particle analysis

434


routines, but MetaMorph will perform semi-automated single particle track-ing with subpixel resolution (15) for bright-field, DIC, and fluorescence images.The centroid data can be output to an Excel spread sheet or to custom-ized programs (9, 13) for assembly of trajectories and calculation of diffusioncoefficients.

References1. DeBrabander, M., Nuydens, R., Geuens, G., Moeremans, M., and DeMey, J.

(1985). Cytobios, 43, 273.2. DeBrabander, M., Geerts, H., Nuyens, R., Nuydens, R., and Corneliessen, F.

(1993). In Electronic light microscopy techniques in modern biomedicalmicroscopy (ed. D. Shotton), p. 141. Wiley-Liss, Inc., New York.

3. Geerts, H., DeBrabander, M., Nuydens, R., Geuens, S., Moeremans, M., DeMey,J., et al. (1987). Biophys. J., 52, 775.

4. DeRoe, C., Courtoy, P. J., and Baudhuin, P. (1987). J. Histochem. Cytochem., 35,1191.

5. Lee, G. M., Ishihara, A., and Jacobson, K. A. (1991). Proc. Natl. Acad. Sci. USA,88, 6274.

6. Van den Pol, A. N., Ellisman, M., and Deerinck, T. (1989). In Colloidal gold: prin-ciples, methods and applications, Vol. 1 (ed. M. A. Hayat), p. 452. Academic Press,San Diego.

7. Berg, H. C. (1983). Random walks in biology, p. 5. Princeton University Press,Princeton.

8. Rudnick, J. and Gaspari, G. (1987). Science, 237, 384.9. Lee, G. M., Zhang, F., Ishihara, A., McNeil, C. L., and Jacobson, K. A. (1993). /.

Cell BioL, 120, 25.10. Qian, H., Sheetz, M. P., and Elson, E. L. (1991). Biophys. J., 60, 910.11. Sheetz, M. P., Turney, S., Qian, H., and Elson, E. L. (1989). Nature, 340, 284.12. Sako, Y. and Kusumi, A. (1994). J. Cell Biol, 125, 1251.13. Simson, R., Sheets, E. D., and Jacobson, K. (1995). Biophys. J., 69, 989.14. Lee, G. M., Johnstone, B., Jacobson, K., and Caterson, B. (1993). J. Cell Biol, 123,

1899.15. Gelles, J., Schnapp, B. J., and Sheetz, M. P. (1988). Nature, 331, 450.

435

List of suppliers

Agar Scientific Limited, 66A Cambridge Road, Stansted, Essex CM24 8DA,UK.

Aldrich Chemical Co., The Old Brickyard, New Road, Gillingham, Dorset,SP8 4BR, UK.

Alfa AesarAlfa Aesar, Johnson Matthey Catalog Company, 30 Bond Street, Ward Hill,

MA 01835-8099, USA.Alfa Aesar, Johnson Matthey Rare Earth Products, Waterloo Road, Widnes,

Cheshire WA8 0QH, UK.AmershamAmersham International plc., Lincoln Place, Green End, Aylesbury, Bucking-

hamshire HP20 2TP, UK.Amersham Corporation, 2636 South Clearbrook Drive, Arlington Heights, IL

60005, USA. (Distributor for Janssen gold products.)Anderman and Co. Ltd., 145 London Road, Kingston-Upon-Thames, Surrey

KT17 7NH, UK.Astrocam, Cambridge, UK.Bangs Laboratories Inc., Carmel, IN, USA.BDH (see Merck BDH)Beckman InstrumentsBeckman Instruments UK Ltd., Progress Road, Sands Industrial Estate, High

Wycombe, Buckinghamshire HP12 4JL, UK.Beckman Instruments Inc., PO Box 3100, 2500 Harbor Boulevard, Fullerton,

CA 92634, USA.Becton DickinsonBecton Dickinson and Co., 21 Between Towns Road, Cowley, Oxford OX4

3LY, UK.Becton Dickinson and Co., 2 Bridgewater Lane, Lincoln Park, NJ 07035,

USA.Bellco Glass Co., 340 Edrudo Road, Vineland, New Jersey 08360-0117, USA.BioBio 101 Inc., do Stratech Scientific Ltd., 61-63 Dudley Street, Luton,

Bedfordshire LU2 0HP, UK.

Al

List of suppliers

Bio 101 Inc., PO Box 2284, La Jolla, CA 92038-2284, USA.Bio-Rad LaboratoriesBio-Rad Laboratories Ltd., Bio-Rad House, Maylands Avenue, Hemel

Hempstead HP2 7TD, UK.Bio-Rad Laboratories, Division Headquarters, 3300 Regatta Boulevard,

Richmond, CA 94804, USA.BioRad Microscience, Hemel Hempstead, Hertfordshire, UK.BioSoft, Cambridge, UK.BiosynthBiosynth Ag, Rietlistr. 4, Unteres Buriet, PO Box 125, Staad, SG 9422,

Switzerland.Biosynth International Inc., 1665 West Quincy Avenue, Suite 155, Naperville,

IL 60540, USA.Boehringer MannheimBoehringer Mannheim UK (Diagnostics and Biochemicals) Ltd., Bell Lane,

Lewes, East Sussex BN17 1LG, UK.Boehringer Mannheim Corporation, Biochemical Products, 9115 Hague

Road, PO Box 504 Indianopolis, IN 46250-0414, USA.Boehringer Mannheim Biochemica, GmbH, Sandhofer Str. 116, Postfach

310120 D-6800 Ma 31, Germany.British Drug Houses (BDH) Ltd., Poole, Dorset, UK.CalbiochemCalbiochem Novabiochem (UK) Ltd., Highfield Science Park, Nottingham

NG7, UK.Calbiochem Corporation, PO Box 12087, San Diego, California, USA.Citifluor Products, The Chemical Laboratory, The University of Kent,

Canterbury CT2 7NH, UK.Clark Electromedical Instruments, PO Box 8, Pangbourne, Reading RG8

7HU, UK.Collaborative Biomedical ProductsCollaborative Biomedical Products, Two Oak Park, Bedford, MA 01730, USA.Collaborative Biomedical Products, do Becton Dickinson UK Ltd., Between

Towns Road, Cowley, Oxford OX4 3LY, UK.Corel Corporation, Ottawa, Ontario, Canada.Dako Ltd., 16 Manor Courtyard, Hughenden Avenue, High Wycombe HP13

5RE, UK.Data Cell Ltd., Hattori House, Vanwall Business Park, Maidenhead, Berkshire

SL6 4UB, UK.Data Translation, 100 Locke Drive, Marlboro, Massacheusetts 01752-1192,

USA.Decon Laboratories Ltd., Conway Street, Hove, East Sussex BN3 3LY, UK.Difco LaboratoriesDifco Laboratories Ltd., PO Box 14B, Central Avenue, West Molesey, Surrey

KT8 2SE, UK.

438

List of suppliers

Difco Laboratories, PO Box 331058, Detroit, MI 48232-7058, USA.Ditric Optics Inc., Hudson, MA, USA.Du PontDupont (UK) Ltd. (Industrial Products Division), Wedgwood Way, Steve-

nage, Hertfordshire SG1 4Q, UK.Du Pont Co. (Biotechnology Systems Division), PO Box 80024, Wilmington,

DE 19880-002, USA.Ealing Electro-Optics plc., Watford, Hertfordshire, UK.EppendorfEppendorf-Netheler-Hinz GmbH, 22331 Hamburg, Germany.Eppendorf, c/o Merck Ltd., Merck House, Seldown Road, Poole, Dorset

BH15 1TD, UK.European Collection of Animal Cell Culture, Division of Biologies, PHLS

Centre for Applied Microbiology and Research, Porton Down, Salisbury,Wiltshire SP4 0JG, UK.

E-Y Laboratories Inc., 107 North Amphlett Blvd., San Mateo, CA 94401,USA.

Falcon (Falcon is a registered trademark of Becton Dickinson and Co.)Fisher Scientific Co., 711 Forbest Avenue, Pittsburgh, PA 15219-4785, USA.Flow Laboratories, Woodcock Hill, Harefield Road, Rickmansworth, Hert-

fordshire WD3 1PQ, UK.FlukaFluka-ChemieAG, CH-9470, Buchs, Switzerland.Fluka Chemicals Ltd., The Old Brickyard, New Road, Gillingham, Dorset

SP8 4JL, UK.Foster Findlay Associates Ltd., Newcastle Technopole, Kings Manor, New-

castle upon Tyne NE1 6PA, UK.Gibco BRLGibco BRL (Life Technologies Ltd.), Trident House, Renfrew Road, Paisley

PA3 4EF, UK.Gibco BRL (Life Technologies Inc.), 3175 Staler Road, Grand Island, NY

14072-0068, USA.Glen Spectra Ltd., Stanmore, Middlesex, England.Graticules Division (see Pyser SGI).Hamamatsu PhotonicsHamamatsu Photonic Systems, 360 Foothill Road, Box 6910, Bridgewater, NJ

08807-0910, USA.Hamamatsu Photonics, Lough Point, 2 Gladbeck Way, Windmill Hill, Enfield,

Middlesex EN2 7JA, UK.Arnold R. Horwell, 73 Maygrove Road, West Hampstead, London NW6

2BP, UK.HybaidHybaid Ltd., 111-113 Waldegrave Road, Teddington, Middlesex TW11 8LL,

UK.

439

List of suppliers

Hybaid, National Labnet Corporation, PO Box 841, Woodbridge, NJ 07095,USA.

HyClone Laboratories, 1725 South HyClone Road, Logan, UT 84321,USA.

ICN Biomedicals Inc., 1263 South Chillicothe Road, Aurora, Ohio 44202,USA.

Imaging Associates Ltd., 8 Thame Park Business Centre, Wenman Road,Thame, Oxon OX9 3XA, UK.

International Biotechnologies Inc., 25 Science Park, New Haven, Connecticut06535, USA.

Invitrogen CorporationInvitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA 92008, USA.Invitrogen Corporation, Invitrogen B.V., PO Box 2312, 9704 CH, Groninen,

The Netherlands.Jackson Immunoresearch Laboratories Inc., 872 West Baltimore Pike, PO

Box 9, West Grove, PA 19390, USA.JASC Inc., Eden Prairie, MN, USA.Kodak: Eastman Fine Chemicals, 343 State Street, Rochester, NY, USA.Leica UK Ltd., Davy Avenue, Knowlhill, Milton Keynes MK5 8LB, UK.Life Technologies Inc., 8451 Helgerman Court, Gaithersburg, MN 20877,

USA.Media Cybernetics, 8484 Georgia Avenue, Silver Spring, Maryland 20910,

USA.MerckMerck Industries Inc., 5 Skyline Drive, Nawthorne, NY 10532, USA.Merck, Frankfurter Strasse, 250, Postfach 4119, D-64293, Germany.Merck BDH, Merck House, Poole, Dorset BH15 1TO, UK.Micro Instruments Ltd., 18, Hanborough Business Park Lodge, Freeland,

Oxford, UK.MilliporeMillipore (UK) Ltd., The Boulevard, Blackmoor Lane, Watford, Hertford-

shire WD1 8YW, UK.Millipore Corp./Biosearch, PO Box 255, 80 Ashby Road, Bedford, MA 01730,

USA.Molecular Probes, Eugene, OR, USA.Molecular Probes Europe BV, Leiden, The Netherlands.NarishigeNarishige Europe Ltd., Unit 7, Willow Business Park, Willow Way, London

SE26 4QP, UK.Narishige USA Inc., 404 Glen Cove Avenue, Sea Cliff, New York 11579,

USA.National Diagnostics Ltd., Unit 4, Fleet Business Park, Itlings Lane, Hessle,

Hull HU13 9LX, UK.New England Biolabs (NBL)

440

List of suppliers

New England Biolabs (NBL), 32 Tozer Road, Beverley, MA 01915-5510,USA.

New England Biolabs (NBL), c/o CP Labs Ltd., PO Box 22, Bishops Stort-ford, Hertfordshire CM23 3DH, UK.

Nicholson Precision Scientific, 7851 Beechcraft Avenue, Gaithersburg, MD29879, USA.

Nikon Corporation, Fuji Building, 2-3 Marunouchi 3-chome, Chiyoda-ku,Tokyo, Japan.

Nyegaard AS, Postbox 4220, Torshov, Oslo 4, Norway.Omega Optical Inc., Brattleboro, Vermont, USA.Optimas Corporation UK, 40 Churchill Square, Kings Hill, East Mailing,

Kent ME19 6DU, UK.Oriel, Stratford, CT, USA.Oxoid Ltd., Wade Road, Basingstoke, Hampshire RG24 0PW, UK.Ted Pella Inc., 4595 Mountain Lakes Blvd. Redding, CA 96003, USA.Perkin-ElmerPerkin-Elmer Ltd., Maxwell Road, Beaconsfield, Buckinghamshire HP9

1QA, UK.Perkin Elmer Ltd., Post Office Lane, Beaconsfield, Buckinghamshire HP9

1QA, UK.Perkin Elmer-Cetus (The Perkin-Elmer Corporation), 761 Main Avenue,

Norwalk, CT 0689, USA.Pharmacia Biotech Europe, Procordia EuroCentre, Rue de la Fuse-e 62, B-

1130 Brussels, Belgium.Pharmacia BiosystemsPharmacia Biosystems Ltd. (Biotechnology Division), Davy Avenue, Knowl-

hill, Milton Keynes MK5 8PH, UK.Pharmacia LKB Biotechnology AB, Bjorngatan 30, S-75182 Uppsala,

Sweden.Photometries Inc., 3440 East Britannia Drive, Tucson, AZ 85706, USA.Polysciences Inc., 400 Valley Road, Warrington, PA 18976-2590, USA.PromegaPromega Ltd., Delta House, Enterprise Road, Chilworth Research Centre,

Southampton, UK.Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711-5399,

USA.Pyser SGI, Morley Road, Tonbridge, Kent TN1 1RN, UK.QiagenQiagen Inc., c/o Hybaid, 111-113 Waldegrave Road, Teddington, Middlesex

TW11 8LL, UK.Qiagen Inc., 9259 Eton Avenue, Chatsworth, CA 91311, USA.Rolyn Optics, 706 Arrow Grand Circle, Covina, CA 91722-2199, USA.Schleicher and SchuellSchleicher and Schuell Inc., Keene, NH 03431A, USA.

441

List of suppliers

Schleicher and Schuell Inc., D-3354 Dassel, Germany.Schleicher and Schuell Inc., do Andermann and Co. Ltd.Shandon Scientific Ltd., Chadwick Road, Astmoor, Runcorn, Cheshire WA7

1PR, UK.Sigma Chemical CompanySigma Chemical Company (UK), Fancy Road, Poole, Dorset BH17 7NH, UK.Sigma Chemical Company, 3050 Spruce Street, PO Box 14508, St. Louis, MO

63178-9916, USA.Sorvall DuPont Company, Biotechnology Division, PO Box 80022, Wilmington,

DE 19880-0022, USA.Spectrum Medical Industries Inc., 1100 Rankin Road, Houston, TX 77073,

USA.StratageneStratagem Ltd., Unit 140, Cambridge Innovation Centre, Milton Road, Cam-

bridge CB4 4FG, UK.Stratagene Inc., 11011 North Torrey Pines Road, La Jolla, CA 92037, USA.Synoptics Ltd., 271 Cambridge Science Park, Milton Road, Cambridge CB8

4WE, UK.Taab Laboratories Equipment Ltd., 3 Minerva House, Calleva Industrial

Park, Aldermaston, Berkshire RG7 4QW, UK.The Binding SiteThe Binding Site Ltd., PO Box 4073, Birmingham B29 6AT, UK.The Binding Site Inc., 5889 Oberlin Drive, Suite 101, San Diego, CA 92121,

USA.United States Biochemical, PO Box 22400, Cleveland, OH 44122, USA.Universal Imaging Corporation, 502 Brandywine Parkway, West Chester, PA

19380, USA.Vector LaboratoriesVector Laboratories Ltd., 16 Wulfric Square, Bretton, Peterborough PE3

8RF, UK.Vector Laboratories, Inc., 30 Ingold Road, Burlingame, CA 94010, USA.Wellcome Reagents, Langley Court, Beckenham, Kent BR3 3BS, UK.Wilmad Glass, US Route 40 and Oak Road, Buena, NJ 08310, USA.Yakult Honsha Co. Ltd., 1-1-19 Higashi-Shinbashi, Minato-ku, Tokyo 105,

Japan.Yakutt Pharmaceutical Ind. Co. Ltd., 1-19 Higashi Shinbashi, Minato-ku,

Tokyo 16, Japan.Carl Zeiss (Oberkochen) Ltd., PO Box 78, Woodfield Road, Welwyn Garden

City, Hertfordshire AL7 1LU, UK.Zymed Laboratories Inc., 52 South Linden Avenue, Suite 3, South San

Francisco, CA 94080, USA.

442

A2

Suppliers for specialist items

Adobe Systems, Inc., 345 Park Avenue, San Jose, CA 95110-2704, USA.AVT GmbH, Benzstrasse 2a, D-63741 Aschaffenburg, Germany.Barco International, Noordlaan 5, B-S520 Kuurne, Belgium.Bio-Rad Microscience Ltd., Hemel Hempstead, Hertfordshire HP2 7TD, UK.Bitplane AG, Technoparkstrasse 1, CH-8005 Zurich, Switzerland.Cargille Laboratories, Inc., 55 Commerce Road, Cedar Grove, NJ 07009-

1289, USA.Carl Zeiss Jena GmbH, Produktbereich Mikroskopie, D-07740 Jena,

Germany.Carolina Biological Supply Comp., 2700 York Road, Burlington, NC 27215,

USA.Clay Adams Co., Parsippany, NJ, USA.COHU, Inc., 5755 Kearny Villa Road, San Diego, CA 92123-5623, USA.Colorado Video, Inc., Boulder, CO 80306, USA.DAGE-MTI, Inc., 701 North Roeske Avenue, Michigan City, IN 46360, USA.Data Translation, Inc., 100 Locke Drive, Marlboro, MA 01752-1192, USA.Datacube, Inc., 300 Rosewood Drive, Danvers, MA 01923, USA.Diagnostic Instruments, Inc., 6540 Burroughs Street, Sterling Height, Michigan

48314-2133, USA.Eastman Kodak Company, 343 State Street, Rochester, NY 14650-2010,

USA.EOS Electronics AV Ltd., Barry, South Glamorgan, UK.FAST Electronic GmbH, Landsberger Strasse, 76, D-80007 Munich,

Germany.For-A Company Ltd., Tokyo 160, Japan.Hamamatsu Photonics Deutschland GmbH, Arzbergerstrasse 10, D-82211

Hersching, Germany.HaSoTec GmbH, Burgwall 20, D-18055 Rostock, Germany.Imaging Technology, Inc., 55 Middlesex Turnpike, Bedford, MA 01730, USA.Improvision, Inc., Viscount Centre II, University of Warwick Science Park,

Coventry CV4 7HS, UK.Inovision Corp., 6321 Angus Drive, Raleigh, NC 27613, USA.Jasc Software, Inc., 11011 Smetana Road, Minnetonka, MN 55343, USA.


Javelin Systems, 23456 Hawthrone Blvd, Torrance, CA 90505-4716, USA.Kappa Messtechnik GmbH, Kleines Feld 6, D-37130 Gleichen, Germany.Leica Microsystems Holding GmbH, Ernst-Leitz-Strasse, D-35578 Wetzlar,

Germany.Matrox Electronic Systems (UK) Ltd., 6 Cherry Orchard West, Kembrey

Park, Swindon, Wiltshire SN2 6UP, UK.Media Cybernetics, 8484 Georgia Avenue, Silver Spring, MD 20910, USA.Mitec GmbH, Prof. Messerschmidt Strasse 3, D-85579 Neubiberg, Germany.Mitsubishi Corp., 5665 Plaza Drive, Cypress, CA 90630, USA.Modulation Optics, Inc., Greenville, NY 11548, USA.Molecular Probes, Inc., 4849 Pichforde Avenue, Eugene, OR 94702-0469,

USA.Motion Analysis, 3617 Westwind Blvd, Santa Rosa, CA 95403, USA.MS MacroSystem Computer GmbH, Borgacker 2-6, D-58454 Witten,

Germany.NIH, Research Services Branch, Bethesda, MD, USA.Nikon Europe BV, Schipholweg 321, 1170 AE Badhoevedorp, The

Netherlands.O. Kindler GmbH & Co., Ziegelhofstrasse 214, D-79110 Freiburg, Germany.Olympus, Inc., Two Corporate Center Drive, Melville, NY 11747-3157, USA.Optronics, 175 Taft Court, Suite 111, Goleta, CA 93117, USA.PCO Computer Optics GmbH, Ludwigsplatz 4, D-93309 Kelheim, Germany.Photometries Ltd., 3440 East Britannia Drive, Tucson, AZ 35706, USA.PTI (Photon Technology International), Inc., Deer Park Drive, Suite F,

Monmouth Junction, NJ 08852, USA.Photonic Science, Millham, Mountfield, Robertsbridge, East Sussex TN32

5LA, UK.Polaroid Export Europe, Wheathampstead House, Codicote Road, Wheat-

hampstead Hertfordshire AL4 8SF, UK. (CCD cameras, slide makers)Polaroid, 575 Technology Square, 9P, Cambridge, MA 02139, USA. (Polarizers)Princeton Instruments, Inc., 3660 Quakersbridge Road, Trenton, NJ 08619,

USA.Proxitronic GmbH, Robert-Bosch-Strasse 34, D-64625 Bensheim, Germany.Quantel Ltd., Turnpike Road, Newbury, Berkshire RG14 2NE, UK.Reichert/Cambridge Instruments, see LeicaScanalytics, Inc., 8550 Lee Highway, Fairfax, VA 22031-1515, USA.Scion Corp., 82 Worman's Mill Court, Suite H, Frederick, MD 21703, USA.Sharp, Inc., 22-22 Nagike-cho Abeno-ku, Osaka 545, Japan.Sigma Chemical Company, 3050 Spruce Street, St Louis Missouri, USA.Sony Corp., 7-35 Kitashinaggawa 6-chome, Shinagawa, Tokyo 141, Japan.Stemmer Imaging GmbH, Gutenbergstrasse 11, D-82178 Puchheim, Germany.Tektronix, Inc., 26600 SW Parkway, Wilsonville, OR 97070, USA.Theta Systeme Dr. Tatarczyk GmbH, John F. Kennedy Strasse 9, D-82194

Grobenzell, Germany.

444


Uniblitz, SD10 Vincent Assoc., 1255 University Avenue, Rochester, NY14607, USA.

Universal Imaging Corp., 502 Brandywine Parkway, West Chester, PA 19380,USA.

VayTek, Inc., 305 E Lowe Avenue, PO Box 732, Fairfield, IA 52556, USA.Vital Images, 505 N Fourth Street, Fairfield, Iowa 52556, USA.

445

Index

aberrationschromatic 53-4and point spread function 51-4and refractive index of specimen 53spherical 51-3

Airy disc 13, 14, 50-1, 107, 108Allen video-enhanced contrast (AVEC) 75, 84,

104-5, 107-8applications 117-23interpretation 116-17limitations 117-22setting up microscope 110-16

anaxial illumination, video microscopy 110,118-19

antibodiesconjugation to colloidal gold 429-30monoclonal 187-8polyclonal antisera 187purification 188specificity 188-9storage 189structure 186-7

atomic force microscopy 342cellular substructures 362-3chromosomes 363, 365-6

autofluorescence, induction by glutaraldehyde204

back focal plane, objective 8, 50Beer-Lambert law, and absorption

measurements 132B-glucuronidase, histochemical method 381-2biolistic process, see particle bombardment

calcium ionsproperties of specific dyes 223-7, 230-1quantification in cells 129, 133, 221-74ratio imaging 129, 133, 230-1second messenger in cells 221-2see also ion imaging

camerasCCD 89-91, 93-4, 126-7, 433film 30-3, 180-1SIT 126video 33-8, 89-97video-intensified microscopy (VIM) 91-5,

123-7carbohydrates, identification using lectins 185C-banding of chromosomes, method 162-3CCD cameras

chromosome analysis 182

ion imaging 242for nanovid microscopy 433video microscopy 89-91, 93-4, 126-7

cell culturefor chromosome studies 152-5, 157mammalian cells 152-5for nanovid microscopy 430-1

cell proliferation, and nucleolus organizingregions (NORs) 338

cell-substrate contacts, visualization 420, 422chromosome banding

AgNOR-banding method 164, 166-7C-banding method 162-3classification 161-2definition 161G-banding method 164kinetochore staining 166-8observation 160, 178

chromosome preparationcytocentrifugation 155-6for immunocytochemistry 155-6mammalian cells 152-5plant cells 156-9quality assessment 159-60

chromosomesatomic force microscopy 363, 365-6fixation 154-9, 167fluorescence microscopy 151, 158, 160-1,

178-81near-field scanning optical microscopy

(NSOM) 363-7observation methods 160, 177-83photomicrography 180-1preparation methods 151-60shear force microscopy 366-8uniform (solid) staining 160-1

colloidal goldconjugation of antibodies 429-30label for immunohistochemistry 197, 209-10nanovid microscopy 425-35reflection-contrast microscopy 280silver enhancement 209-10

condensersdark-field illumination 21light microscope 2-4, 7, 10

confocal microscopy 45-71choice of objectives 64—5chromosome analysis 181-2comparison with other techniques 58-61,

182,422ion imaging 243noise 58-60, 61optical sectioning 62-3

Index

confocal microscopy (contd.)out-of-focus light 60-2photobleaching 242point scanning 54-5and point spread function 48-51, 246-8principle 47-8setting up 68-70slit scanning 55-6specimen preparation 63-8spinning (Nipkow) disc 56-7two-photon imaging 57-8ultraviolet 54use of 62-70

conjugate planes, light microscope 7-10contrast methods

bright-field 16-17, 40-1dark-field 17-23, 40-1, 117-18differential interference contrast (DIC)

25-6, 40, 77fluorescence microscopy 26, 41, 45Hoffman modulation contrast 110, 118nanovid microscopy 425-35phase-contrast 22-3, 40polarized light 23-8, 40Rheinberg illumination 17test specimen 38-41video-enhanced contrast (VEC) 73, 75, 77-8,

106-8video-intensified microscopy 29, 40, 75-8,

123-7coverslips

sealing 109-10thickness 2, 108-9, 246

culture chambers, for living specimens 248cytocentrifugation, chromosome preparation

155-6

dark ground contrast, see contrast methods,dark-field

diatoms, test specimens for resolution 10-12differential interference contrast (DIC) 25-6,

40,77observation of chromosomes 160, 178and video microscopy 107, 110-11, 113,

115-16, 120-2dry mass measurement, interference

microscopy 39

electron microscopy, and reflection-contrastmicroscopy 298

electrophysiology, and ion imaging 270electroporation, introduction of dyes into cells

235-6embedding

paraffin for light microscopy 313-14resin 301-5

evanescent illumination, see total internalreflection fluorescence (TIRF)

extracellular matrix, study by nanovidmicroscopy 428

eyepieceslight microscope 2, 6-9Ramsden disc 6, 9

fading of fluorochromes, see photobleachingfield of view, light microscope 2—3filters

fluorescence microscopy 179, 239-40light microscopy 16-18, 32-3, 178, 180

fixationchromosomes 154-9, 167glutaraldehyde 64, 301-4for histomorphometry 313-14for immunohistochemistry 167, 189-91,

300-4paraformaldehyde 65-6

fixatives, formulae 190-1fluorescence

measurement using video microscopy 132-3standards 246-8video microscopy 121-2

fluorescence lifetime imaging 422fluorescence microscopy 26, 41, 45

antifade mountants 65-6, 175-6, 179-80applications 45-6choice of objectives 64-5, 178chromosomes 151, 158, 160-1, 178-81comparison with confocal microscopy 58-61epi-illumination 45,178-9filters 179, 239-40illumination 178-9, 239of ions in cells 237-45photobleaching 57, 69, 179-80, 223, 226-9, 242setting up 245-8signal-to-noise ratio 254-6

fluorescence recovery after photobleaching(FRAP) 129

fluorescence in situ hybridization (FISH), see insitu hybridization

fluorescent analogue cytochemistry 127-9fluorochromes

calcium-sensitive 222-7, 230-1for chromosome studies 179excitation and emission wavelengths 179photobleaching 57, 69, 179-80, 226-9pH-sensitive 222-5, 228-31

G-banding of chromosomes, method 164glutaraldehyde

fixation 64, 301-4induction of autofluorescence 204

green fluorescent protein 46, 380, 385-6, 395

448

Index

haematoxylin and eosin, staining method 314-15histomorphometry 131-2, 311-40

anisotropy 326colour imaging 334image analysis 327-38nuclear volume measurement 321-3, 329, 336shape measurement 337-8specimen preparation 313-18statistical analysis 326-7volume fraction measurement 323-6, 337

Hoffman modulation contrast 110, 118horseradish peroxidase

label for immunohistochemistry 196-7,204-7, 214, 317-18

silver enhancement 205-6

illuminationcolour temperature 32fluorescence microscopy 178-9, 239ion imaging 239K6hler 7-10, 31light microscope 1-4,7-10reflection-contrast microscopy 282-3two-photon microscopy 57-8, 244video microscopy 101-2,112-13

image formationlight microscope 3-6reflection-contrast microscopy 286-90

image processinganalogue 75, 95-7chromosome analysis 183dangers 183digital 75, 97-101histomorphometry 327-38ion imaging 249, 267for nanovid microscopy 433-4video microscopy 78-89, 95-101

image recordingdrawing 29-30photomicrography 30-3, 38, 180-1video cameras 33-7, 38, 73-149see also photomicrography

immersion oil, choice of 109immunocytochemistry, see

immunohistochemistryimmunoglobulins, see antibodiesimmunohistochemistry 185—220

adhesion of sections to slides 210-12of cell smears 215—18chromosome preparation 155-6controls 213double labelling 214-15fixation for 167, 189-91kinetochores 166-8labelling methods 202-13, 218-19labels 194-201, 204-10, 214, 317-18, 429-30mountants 204, 212-13

problem solving 213-14quantification 218reflection-contrast microscopy 275-6, 280,

288, 290-1, 295, 305-8resin sections 305-7specimen preparation 189-94, 298-301unmasking hidden antigens 192-4

Inoue' system, video-enhanced contrast (VEC)107-8

in situ hybridizationapplications to chromosomes 168-70labels 171-3method 173-7near-field scanning optical microscopy

(NSOM) 365-8probe preparation 170-3reflection-contrast microscopy 275-6, 295-6resolution 170

interference microscopydry mass measurement 39measurement of optical path differences 29see also differential interference contrast

(DIC)internal reflection microscopy

comparison with total internal reflectionfluorescence (TIRF) 422

visualization of cell-substrate contacts 422introduction of materials into living cells, see

electroporation; ion-sensitive dyes;microinjection; particle bombardment

ion imagingcalcium measurement in cells 221-74calibration 253, 260-4combination with other techniques 269-70confocal microscopy 243controls 270-1and electrophysiology 270equipment for 240-5filters 239-40fluorochromes for 222-31image processing 249, 267light sources 239multiphoton imaging 244-5multiple detectors 245pH measurement in cells 221-74quantification 252-67ratio imaging 249-52ratiometric dyes 230-1signal-to-noise ratio 254-6statistical analysis 264-7

ion-sensitive dyesintroduction into cells 231-7photobleaching 223, 226-7, 242, 263

kinetochores, immunocytochemical labelling166-8

Kohler illumination 7-10, 31

449

Index

lectins, identification of carbohydrates 185light, interactions with matter 5-6light microscope

condensers 2-4, 7, 10conjugate planes 7-10eyepieces 2, 6-9field of view 2-3illumination 1-4, 7-10, 31image formation 3-6linear measurement 319-20magnification changer 3, 105, 106objectives 2-6, 9-14, 64-5, 102-4resolution 6, 10-14, 45setting up 7-10, 318-19

light sources, see illuminationlinear measurement, light microscope 319-20living specimens

culture chambers 248effects of irradiation 248-9observation 109, 110, 248reflection-contrast microscopy 275, 289, 308specimen preparation 108-9temperature control 249

luminescence 129

magnification, empty 10-11, 14-15magnification changer, light microscope 3, 105,

106mammalian cells

chromosome preparation 152-5culture 152-5

membrane mobility, study by nanovidmicroscopy 428, 434-5

microinjectionadvantages 386-7equipment 387-91identification of cells 391-2, 394-5introduction of materials into living cells

386-95technique 392-4and total internal reflection fluorescence

(TIRF)410, 412microspectrofluorimetry 129molecular imaging 129monolayers, near-field scanning optical

microscopy (NSOM) 358-62mountants

antifading for fluorescence microscopy 65-6,175-6, 179-80

for immunohistochemistry 204, 212-13reflection-contrast microscopy 307

movement, detection by video microscopy87-8, 101, 122-3, 131-2

nanovid microscopy 425-35analysis of mobility in membranes 428, 434-5

camera 433cell culture 430-1image processing 433-4image storage 434microscope 432-3observation of colloidal gold particles

425-35principle 425-7study of extracellular matrix 428viewing cells 431-2

near-field scanning optical microscopy(NSOM) 341-71

applications 344, 355-67chromosomes 363-7distance regulation 349-50in situ hybridization 365-8instrumentation 344-9monolayers 358-62operation 353-4principle 342-3probe design 344-9probe fabrication 351-3resolution 346, 355, 359, 362-3, 367, 369and shear force microscopy 349-50, 353-4single molecule detection 355-8subcellular structures 362-3viruses 362

neurobiology, applications of videomicroscopy 130

neurons, visualization by video-enhancedcontrast 130

Nomarski interference, see differentialinterference contrast (DIC)

nucleolus organizing regions (NORs)measure of cell proliferation 338silver staining 164, 166, 315-16

numerical apertureand light-gathering power 102-3objectives 2, 6, 10-14, 50, 102-3and resolution 6, 10-14, 45

objectivesback focal plane 8, 50cleaning 114, 246for confocal microscopy 64—5coverslip thickness 246for fluorescence 64-5, 178immersion 2, 53, 64—5light-gathering power 102-3light microscope 2, 6, 9-14, 64-5, 102-4light transmission 103numerical aperture 2, 6, 10-14, 50, 102-3reflection-contrast microscopy 277-81, 284resolution 6, 10-14water immersion 109, 110

optical microscope, see light microscope

450

Index

out-of-focus lightremoval by confocal microscopy 46, 60-2removal by deconvolution 46, 61

paraformaldehyde, fixation 65-6particle bombardment

apparatus 374-5applications 373identification of transformed cells 380-6introduction of materials into living cells

373-86particle preparation 375-7target tissue 377-80

PHproperties of specific dyes 223-5, 228-31second messenger in cells 221-2

phase-contrast 22-3observation of chromosomes 160, 178video microscopy 119

pH measurementin cells 133, 221-74ratio imaging 133, 230-1see also ion imaging

photobleaching, fluorochromes 57, 69, 179-80,223, 226-9, 242

photography, video microscopy 142-3photomicrography 30-3, 38

black and white 32-3, 180chromosomes 180-1colour 32-3, 180-1

photon scanning tunnelling microscopy(PSTM) 345, 349

plant cells, chromosome preparation 156-9point spread function

and aberrations 51-4and confocal imaging 48-51, 246-8and resolution 246-8

polarized lightcontrast methods 23-8, 40video-enhanced contrast (VEC) 113-14,

119-20,122

quantificationabsorption 132fluorescence 132-3, 237, 252-67immunohistochemistry 218ions in cells 132-3, 230-1, 237, 252-67

Ramsden disc, eyepieces 6, 9ratio imaging

calcium measurement in cells 129-33, 230-1ions in cells 129, 133, 230-1, 249-52, 257-60pH measurement in cells 133, 230-1problems 258-60quantification 257-67

Rayleigh criterion, resolution 13-14, 82reflection-contrast microscopy 121, 275-310

applications 275-6, 290-8colloidal gold 280detection of rare events 296-7and electron microscopy 298focusing 284-6image formation 286-90immunohistochemistry 275-6, 280, 288,

290-1, 295, 305-8in situ hybridization 275-6, 295-6light sources 282—3living cells 275, 289, 308mounting medium 307objectives 277-81, 284optical systems 276-86specimen preparation 290-1, 298-305unstained material 294

refractive index, measurement 39resin sections

immunohistochemistry 305-7specimen preparation 301-5

resolutiondiatoms as test specimens 10-12in situ hybridization 170ion imaging 252—3near-field scanning optical microscopy

(NSOM) 346, 355, 359, 362-3, 367, 369and numerical aperture 6, 10-14objectives 6, 10-14,45photographic film 86photon scanning tunnelling microscopy

(PSTM) 349point spread function 246-8Rayleigh criterion 13-14, 82Sparrow criterion 14, 82star test 13-14video microscopy 75-7, 82, 86

Rheinberg illumination 17RNA transcription, in vitro 67-8, 70

scanning probe microscopy 341-2second messengers 221-2shear force microscopy

cellular substructures 363-4chromosomes 366-8and near-field scanning optical microscopy

(NSOM) 349-50, 353-4signal-to-noise ratio, fluorescence microscopy

254-6silver enhancement

colloidal gold 209-10horseradish peroxidase 205-6

silver staining, nucleolus organizing regions(NORs) 164, 166, 315-16, 338

single molecule detection, near-field scanningoptical microscopy (NSOM) 355-8

451

Index

slides, adhesion of specimens 66, 210-12, 248,307-8

specimen preparationchromosomes 151-60for confocal microscopy 63-8histomorphometry 313-18immunohistochemistry 189-94, 298-301living specimens 108-9reflection-contrast microscopy 290-1,

298-305resin sections 301-5for video microscopy 108-10

specimens, adhesion to slides 66, 210-12, 248,307-8

stainingchromosomes 160-7haematoxylin and eosin 314-15

star test, resolution 13-14statistical analysis

histomorphometry 326-7ion imaging 264-7

stereologydefinition 312symbols 313see also histomorphometry

stray light 79-80, 84, 107-8, 119, 121surface fluorescent microscopy, see total

internal reflection fluorescence (TIRF)surface phenomena, microscopical detection

399-423

time lapse microscopy 137-8, 420total internal reflection fluorescence (TIRF)

applications 399, 420-2combination with other microscopical

techniques 401, 417-18comparison with internal reflection

microscopy 422experimental tips 417-20and microinjection 410, 412microscopical configurations 407-17principles 399-401theory 401-7

transformed cells, identification 380-6two-photon microscopy 57-8, 242, 244-5

illumination 57-8, 244ion imaging 244-5photobleaching 242

ultraviolet microscopy, confocal 54

video camerasimage recording 33-8for nanovid microscopy 433

video-enhanced contrast (VEC) 73, 75-84,106-23

illumination 101-2Inoue system 107-8polarized light 113-14, 119-20, 122setting up the microscope 110-14visualization of neurons 130see also nanovid microscopy

video-intensified microscopy (VIM) 29, 40, 73,75-8, 123-7

applications 127-30cameras 91-5, 123-7image acquisition 124-7microscope requirements 123-4

video microscopy 73-149anaxial illumination 118-19applications 117-23cameras 89-97chromosome analysis 182colour images 127dark-field 117-18detection of movement 87-8, 122-3, 131-2and differential interference contrast 107,

110-11, 113, 115-16,120-2digital image processing 76, 84-9, 97-9editing video tapes 143-6fluorescence 121-2fluorescence measurements 132-3Hoffman modulation contrast 118illumination 101-2, 112-13image intensification 29, 40, 77-9image recording 133-46light sources 101-2microscope requirements 101-6phase-contrast 119printouts for presentation 141-2, 143recording equipment 133-7reflection-contrast 121, 275-310resolution 75-7, 82size measurements 131storage media 140time lapse recording 137-8

viruses, near-field scanning optical microscopy(NSOM) 362

452

light microscopy in biology: a practical approach (the practical approach series) (2nd edition)

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