manual of electron microscope

Electron Microscopy:A Handbook of Techniques for the BiologistPrefaceAcknowledgments

INTRODUCTION TO ELECTRON MICROSCOPY

UNIT 1 - PREPARATION OF BIOLOGICAL SAMPLES FOR TEMChapter 1 - Chemical FixationChapter 2 - Ultrathin Sectioning & UltramicrotomyChapter 3 - Post-StainingChapter 4 - Grids & Grid Supports

UNIT 2 - PREPARATION OF BIOLOGICAL SAMPLES FOR SEMChapter 5 - Hard Tissue PreparationChapter 6 - Soft Tissue PreparationChapter 7 - Alternative SEM Specimen Preparation

UNIT 3 - BLACK & WHITE PHOTOGRAPHIC PRINCIPLES...Chapter 8 - Film and Paper CompositionChapter 9 - Processing (Films and Papers)Chapter 10 - Negative Handling and Exposure (TEM & SEM)Chapter 11 - Enlargement Printing

Stephen J. BeckNassau Community College

A Handbookof Techniques

for the Biologist

ElectronMicroscopy:

Electron Microscopy:A Handbookof Techniquesfor the Biologist

Electron Microscopy:A Handbook of Techniques

for the Biologist

Stephen J. BeckNassau Community College

CONTENTS

v

TABLE OF CONTENTS

Preface viiAcknowledgments ix

INTRODUCTION TO ELECTRON MICROSCOPY 1

UNIT 1 - PREPARATION OF BIOLOGICAL SAMPLES FOR TEM 6Chapter 1 - Chemical Fixation 6

Additives 10Fixation Times & Temperatures 10Preparation of Fixatives 11Methods of Fixation 15“Routine” Biological Soft Tissue Protocol 16Tissue Processing Note 19Embedding Media 20Fixation Schedule 23Fixation Schedule Worksheet 24Final Chemical Fixation Considerations 25

Chapter 2 - Ultrathin Sectioning & Ultramicrotomy 26Processing of Embedded Blocks - Block Trimming 26Glass Knife Making 29Diamond Knives 33Ultramicrotomy 34

MT-2B Ultramicrotome Sectioning Procedure 39Troubleshooting Guide to Ultramicrotomy 44Materials Required for Ultrathin Sectioning 46

Chapter 3 - Post-Staining 48Uranyl acetate 48Lead Citrate 49Post-staining Procedure 49

Chapter 4 - Grids & Grid Supports 52Grids 52Grid Supports 52

Formvar Films 53Carbon Coating 56

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CONTENTSUNIT 2 - PREPARATION OF BIOLOGICAL SAMPLES FOR SEM 58

Chapter 5 - Hard Tissue Preparation 59

Chapter 6 - Soft Tissue Preparation 60Critical Point Dryer Operation 63Alternatives to Critical Point Drying 67

Fluorocarbon Drying 67Organo-Silicon Compounds 67

Conductive Coating 68Vacuum Evaporation 69Sputter Coater 69Denton Desk II Operation 71

Comparison of TEM and SEM Soft Tissue Protocols 73Fixation Schedule 74Fixation Schedule Worksheet 75

Chapter 7 - Alternative SEM Specimen Preparation 76Uncoated Specimens 76Cryofracture Technique 77Microbial Specimen Preparation 79

Microbial Fixation Schedule Worksheet 83

UNIT 3 - BLACK & WHITE PHOTOGRAPHIC PRINCIPLES... 84Chapter 8 - Film and Paper Composition 85

Emulsion 86Film Speed (ISO/ASA) 86Supports (Base) 87Routine Photographic Films and Papers Used For TEM and SEM 88

Chapter 9 - Processing (Films and Papers) 89Development 90Stop Bath 91Fixer 92Washing 93Drying 94

Chapter 10 - Negative Handling and Exposure (TEM & SEM) 95TEM - Hitachi HS-8 95HS-8 Camera System and Film Exposure 96SEM - Hitachi S-2400 98S-2400 Camera System and Film Exposure 99

Chapter 11 - Enlargement Printing 102Photographic Paper Grades 102Process of Enlargement Printing 103Enlargement Printing Variables 104Printing Tricks 105

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vii

PREFACE

The development of electron optics has had a profound effect on the study of life; its structure andfunction. Without the high technology tools known as the transmission (TEM) and scanning (SEM)electron microscopes, we would understand little about the world of the cell. As proof, I offer anytypical college level introductory and advanced biology course, the textbooks these courses utilize, andany number of biological periodicals/journals. Biology professors will lecture on a variety of cellularstructures and their related functions. Textbooks at all levels are crammed with TEM and SEMphotomicrographs in an effort to illustrate any number of rudimentary concepts. Even to this day, cuttingedge research relies on high resolution images as can be seen in many biological publications. In today’shigh technology environment, the tools of scientific inquiry are easily overlooked and often taken forgranted. Of course, one must understand that these instruments are tools; a means to an end. It willrequire a scientist to make sense of the images presented, someone who understands how the imageswere produced, even to the point of anticipation of a given result.

The novice in electron optics must first learn technique and the theory behind it. The purpose of thishandbook is to provide a detailed explanation and procedural guide to the many tedious procedures ofbiological electron microscopy, TEM and SEM. It can be used in the laboratory as a step by step guideand outside of the laboratory as the student attempts to comprehend the many concepts of biologicalelectron optics. This handbook is intended for introductory college level courses in TEM and SEM.Depending on the college, this could mean the undergraduate (two and four year institutions) or even thegraduate level. At Nassau Community College, the specific relevant courses are Transmission ElectronMicroscopy (BIO 221) and Scanning Electron Microscopy (BIO 222). In my ten years of teaching thesecourses at NCC, I have had the pleasure to introduce this valuable discipline to students with variedbackgrounds and experiences, from traditional two year college students to those with earned Ph.D.’s.Regardless of prior education, students begin the EM courses at essentially the same level. Thishandbook was created to assist any individual in the attainment EM skills so that they will be able toutilize these important tools as they strive to answer the questions of life processes.

The book is divided into three main units; Unit 1 covers the many topics of TEM biological samplepreparation and is an excellent starting point in your understanding of biological EM. Many later topicsin the book refer back to concepts introduced in this unit. Unit 2 involves the preparation of biologicalsamples for the SEM. Even if you are only taking a course in SEM, much of the chemistry of chemicalfixation is found in Unit 1, therefore, the SEM student is urged to refer back to the pertinent points. AtNCC the ideal course sequence begins with the TEM course and is followed by the SEM course, whichis logic I have followed in organizing this handbook. The final Unit 3 covers the concepts of black andwhite photography relative to both TEM and SEM. Here you will find general concepts of silver basedphotography followed by specific treatments of both TEM and SEM photomicrography and imagecapture using the Hitachi HS-8 TEM and the Hitachi S-2400 SEM, both of which are easily related toother common instruments in use today.

As you progress in your education, I expect that these skills you will acquire and the discipline that ittakes to master them will serve you well, whether you actually use electron microscopy in the future ornot.

S.J.B.August, 1996

viii

ACKNOWLEDGMENTS

I would like to thank a number of individuals who have helped to make this book possible. Firstly, mywife and lifelong partner Mary and my children, Jonathan, Bradley and Jessica, for allowing me theprecious time to write the manuscript. To my parents, Jack and Mary for providing me with theopportunity for an education and serving as such positive role models.

In addition, I acknowledge the influence of my mentors, Dr. Kenneth Erb and Dr. Gary W. Grimes, bothof Hofstra University. As my graduate advisor, Ken Erb guided my development as a scientist capableof conducting original research. I must confess that most of the information in this manual is taken fromthe electron microscopy experiences provided by Gary Grimes, a true expert and innovator in the fieldof electron microscopy.

Finally, I would like to thank any of the faculty of Nassau Community College who have supported myendeavors over the past ten years, especially Dr. Dudley Chin, who as biology department chair, alwaysencouraged even my failed attempts. His vision has initiated the technological revolution in thedepartment as we approach the turn of the century. In addition, I thank Dr. Baruch May and Dr. PatriciaCassin, who as co-authors of two successful NSF grants, have brought an awareness of technologicalinnovation to the college and our students. I also acknowledge the 1993/94 NCC Sabbatical Committeemembers who approved the sabbatical which made it possible for me to write the bulk of thismanuscript. I finally acknowledge the support and foresight of the NCC administration, President SeanFanelli and Vice President Jack Ostling for their continuing support of electron optics and other hightechnology endeavors at the college.

ix


Introduction to Electron Microscopy

When Max Knoll and Ernst Ruska were designing the original Transmission ElectronMicroscope (TEM) in Germany in the late 1920’s, they envisioned no biologicalapplications for their instrument, provided it would even function as theoreticallyconceived. Today, the impact of electron optics, Transmission (TEM), Scanning (SEM), anda variety of others, is apparent. Our ability to directly resolve the microanatomy of the cellusing these instruments has revolutionized our very understanding of life and its requisiteprocesses. We take it for granted when a classroom instructor or a textbook describes thecristae of a mitochondrion, the 9+2 ciliary microtubule arrangement, ribosomal subunitsand the phospholipid bilayer of a unit membrane. Where many cellular processes havebeen elucidated using a biochemical “grind & spin” approach (cell homogenization and cellfractionation via ultracentrifugation) followed by characterization of bio-molecules byisolation and purification using techniques such as gel electrophoresis, the cellularbiochemist will often finally desire an image to support their chemical findings. The oldmaxim applies here, “a picture is worth a thousand words”.

Even though the operating environment of the EM is a high vacuum (10-5 Torr) andbiological samples must be preserved or fixed for examination, we have a unique insight tothe molecular architecture responsible for life sustaining functions through the highresolving power of these instruments. Since structure begets function, and the EM canproduces images of fixed cellular ultrastructure, it follows that we can come to a betterunderstanding of cellular physiology through such images/electron micrographs. Of course,it becomes vital to comprehend how electron images are produced and how samples arehandled in order to derive valuable data from a micrograph. This handbook will providethe novice with the procedural and theoretical information they need in order to makesense of the final product - the electron photomicrograph.

The Transmission Electron Microscope (TEM), as the name implies, transmits a highenergy electron beam through a specimen in a high vacuum environment. The vacuumenvironment is required to prevent electron interactions with air molecules which wouldserve to randomly scatter the electrons. In order for the electron beam to penetrate asample, it must be extremely thin, approximately 600-900Å thick. Most samples will haveto be sectioned using an instrument known as an ultramicrotome. Soft tissues will need tobe mechanically strengthened by epoxy resin embedment to withstand the forces ofcutting.

The TEM can be viewed as an inverted light microscope (LM), with the source at the top ofthe instrument. The source of the TEM is a high voltage gun (50,000 volts or higher) whichcontains a pointed tungsten hairpin filament across which the high potential (voltage)is applied. The filament is housed in a metal cylinder with a central aperture (circularopening). This cylinder is known as the Wehnelt cylinder or Bias Cap since it is held at aslightly negative potential (the “bias”) with respect to the filament. This causes initialelectrostatic repulsion of electrons coming off of the filament and the saturation of theWehnelt cylinder. An anode, also with a central aperture, is held at ground potential. Due

ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST2

to the large potential difference between the cathode, now saturated with electrons, andthe anode, the electrons are accelerated through the distance between these two points -this is known as the accelerating voltage. Wide-angle electrons will be grounded outand some will continue at high velocity through the anode aperture in order to contributeto image formation. The electrons will continue to the first lens in the TEM, thecondenser. This lens will refract or bend the source electrons to the specimen. It shouldbe noted that the lenses of an electron microscope are electromagnetic consisting of an ironshroud with a central bore and external copper windings. By varying the current throughthe copper windings, the focal point of the lens can be modified. This effect can allow foradjustments to brightness, magnification and focus.

Once the electrons are focussed by the condenser lens, they will encounter the ultrathinspecimen which is mounted on a grid and placed in a finely machined mechanical stage(with fine micrometer X,Y movements). Based on the electron density of various regions ofthe sample, some electrons will be backscattered while others will continue to transmitthrough the specimen. These transmitted electrons will next encounter the short focallength objective lens assembly which serves as the main imaging lens in the TEM and iscritical to final magnification and focus of the image. This region of the TEM also includesan electromagnetic stigmator which is designed to reshape objective lens asymmetryarising from imperfect lens bores and lens and aperture contamination. Astigmatism(stigma meaning spot) results in stretched images of poor resolution. The octupole (8-pin)stigmator reshapes lens asymmetry by creating an asymmetric elliptical field to counterthe lens distortion. The electron beam finally passes through a magnifying/demagnifyingintermediate lens and then a magnifying projector lens which also functions to projectthe final real image on a fluorescent viewing screen. Since humans are not sensitive toelectrons, the fluorescent material of the view screen is necessary to form an image thatwe can see. Electrons which pass through the electron transparent regions of the specimenwill strike the fluorescent material causing it to emit photons which our eyes are sensitiveto. Such region will appear bright. Specimen regions which are stained (usually withheavy metals) are electron dense and will not allow the transmission of electrons. Theywill not come in contact with the fluorescent screen and these regions will appear dark.This provides the contrast vital to image formation. A piece of film can be introduced viaa camera mechanism beneath the view screen and a permanent exposure recorded. Thiswill be explained in Unit 3.

In the TEM, a direct image is formed by the differential absorption of a transmittedelectron beam. Electron dense versus electron transparent specimen regions areultimately responsible for contrast. Resolving power (RP), the ability to distinguish twopoints as two separate and distinct points, is based on the wavelength of the transmittedelectron source. Resolving power and source wavelength are inversely proportional. Aswavelength decreases, the resolving power increases as given by the Abbe equation:

0.61λRP = ————

n (sin α)


where λ is the source wavelength, n is the refractive index of the medium through whichthe source passes and α is one-half the objective lens acceptance angle. The relation n (sinα) is also known as the numerical aperture (NA) of the lens and under most idealsituations is usually equal to approximately 1.0, making the numerator of the equationmost significant. In simple terms, the resolving power is essentially equal toapproximately one-half of the source wavelength. For visible light, the shortestviolet range wavelength is on the order of 400nm (equivalent to 4,000Å or 0.4µm). Giventhe above equation, the highest resolving power attainable using visible light is about0.2µm (bacterial cell range). Electron wavelength is based on the de Broglie relationshipstated as follows:

hλ = ————

mv

where λ is the source wavelength, h is Planck’s constant, m is the mass of the particle(such as an electron) and v is the velocity of the particle. Substituting electron mass andvelocity at one-third the speed of light (achieved using a 50kv electron gun/acceleratingvoltage, the wavelength of the electron is approximately 0.05Å. Given the Abberelationship, the theoretical resolving power of a 50kv TEM would therefore be 0.025Å.Unfortunately, due to lens aberrations (spherical aberration, chromatic aberration andastigmatism) which cannot be totally corrected for in an EM, the actual resolving powerlimit for a modern TEM is about 2Å - easily molecular resolution, approaching the atomiclevel (for example, the naked DNA double helix is 20Å in width). It becomes clear that thehigher the accelerating voltage at the gun, the greater the electron velocity will be and theshorter the electron wavelength leading to a higher resolving power. Most modern TEM’shave maximum accelerating voltages exceeding 100kv.

In order to achieve the highest resolving power, a single constant electron wavelength isrequired otherwise, chromatic aberration will degrade resolution. Voltage stabilizationcircuitry is critical with the absence of a source spectrum and therefore, the absence of“color” images - unless one uses computer enhancements or Dr. Martin’s dyes applieddirectly to photographic prints.

In the year 1938, Knoll and von Ardenne constructed the first Scanning ElectronMicroscope (SEM) prototype. They suggested that secondary electrons could becollected from the tops of opaque surfaces, the resultant signal then amplified and used tomodulate the grid of a cathode ray tube (CRT). It took many years of refinement toproduce a commercial SEM (1963) - the Cambridge Stereoscan. By comparison, the firstcommercial TEM (1938) - the Siemens Elmiskop, was available soon after the initial TEMdevelopment.

At first glance, the SEM appears to have a simpler design than the TEM with a noticeablyshorter column. The TEM and SEM have an identical electron gun/anode design, however,


the SEM maximum voltage is approximately 25-30kv when using a tungsten hairpinfilament. What follows is simply a series of 2-3 condenser lenses which serve to demagnifythe primary electron beam diameter. In the SEM, resolving power (~30-40Å) is dictated bythe diameter of the primary electron beam which scans the specimen surface in a rasterpattern, much like the electron gun(s) in your TV scan the phosphorus pixels (pictureelements) coated on the inside of the picture tube, in order to form an image. The SEMscan generator is responsible for the raster scan of the sample surface by the primaryelectron beam.

When the primary electron beam contacts the sample surface, a variety of energeticphenomena arise which can be detected and collected (provided the SEM is outfitted withthe appropriate detector). The most common type of energetic phenomenon emitted is thesecondary electron signal. This secondary electron signal is collected to form the typical,virtually three-dimensional image that is usually encountered in textbooks andpublications. Other types of signal include backscattered electrons (BSE),characteristic x-rays (which can be used to create an elemental surface map, andphotons or cathodoluminescence. Once again, each require a specific detector which areeasily added to a modern SEM.

The weakly negative secondary electrons are emitted after surface contact with theprimary electron beam. These secondary electrons are collected by a scintillator-photomultiplier detector. The signal is then amplified and routed to the electron gun ofa black & white viewing CRT. Whether the pixels remain dark or light up is related to thelevel of signal arising from a given specimen area. Maximum signal gives rise to a bright/white pixel, whereas, minimum signal results in a dark/black pixel. The final result iscontrast and a black and white image on the viewing CRT. The amount of signal emittedfrom the specimen surface is a function of its topography or relief. High points of relief,in direct line of sight with the detector and primary electron beam, will produce themaximum signal and appear brighter than low lying areas, which will appear dark. Thefinal image is indirect, based on point by point differential contrast due to the yield ofsecondary electrons from the sample surface.

Magnification is a function of the length of a line scanned on the sample as comparedwith the length of a line scanned on the viewing or photo CRT. Since the CRT dimensionsnever change, a magnification increase is effected by simply modifying your scangenerator control to scan a smaller area/shorter line on the sample. Samples are mountedon aluminum stubs which are placed into the finely machined stage of the SEM. The stagehas provisions for movements in the X, Y, Z (also known as working distance) directions,including T (tilt) and R (continuous 360˚ rotation). A benefit of the SEM image is its highdepth of field and focus which leads to the striking, almost three-dimensional images.

In addition to the components described above, a stigmator is required in the design of aSEM and is one of the most difficult adjustments to teach the novice. Once again, the X, Ystigmation is required to compensate for lens asymmetry which arises from lensimperfections and contamination. One must learn to see the image stretching as you go


through fine focus in order to correct it with the stigmator.

The following illustration compares the design of the LM, TEM and SEM:

+ -

Light Microscope (inverted) Transmission Electron Microscope (TEM) Scanning Electron Microscope (SEM)

sourceilluminator/bulb electron gun

condenser lens(focus source at specimen level)

glass: fixedfocal length

electromagnets: variable

focal length viavariation in lens

current

Demagnifies beam

from 50,000 Å to100 ÅTEM double condenser

(increases brightness)

aperture(reduces extraneous source)

irisdiaphragm

condenseraperture

specimenglassslide

grid

objective lens(magnify & focus)

insertion mechanism

objective aperture (TEM)(grounds stray electrons which

increases contrast)

intermediate lens (1-3)(TEM only - magnify)

ocular(s)projector lens

(magnify & focus final real imageon retina of eye [LM] or

fluorescent view screen [TEM])

fluorescent screen (TEM)(direct imaging)

camera(to record permanent, high resolution image)

film film

eyes/retina

+

First Condenser Lens

Second Condenser Lens

Third Condenser Lens

Final Aperture

Detector

MagnificationControl

Signal (secondary electrons)

camera


UNIT 1 - PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

The high vacuum environment (10-5 Torr) of the transmission electron microscopepresents a major and obvious obstacle for the study of biological samples. Since death isthe inevitable result of introducing a living organism into a vacuum environment, we arelimited to the examination of dead specimens. Another problem that prevents the study oflive organisms is the requirement that samples be sectioned ultrathin. Unless samples arethin enough, between 600-900Å, the electron beam cannot transmit or pass through it.Once cut, a cell cannot typically be expected to live. Unfortunately, as a result of these twolimitations, we are not able to study actual processes occurring directly within the cell,even though the TEM has the resolving power to do so.

Chapter 1 - Chemical Fixation

Chemical fixation involves killing and preserving the organism/organ/tissue/cell in as life-like conditions as possible. The biological structures and their functions are fixed or“frozen” in time and space. Our ability to observe fixed biological ultrastructure using theTEM allows us to infer and come to understand function. The true goal of fixation is toenable the investigator to examine the structure(s) they are interested in (to see whatyou want to see!).

Since we now understand that structure and not direct function is studied using the TEM,we must determine the major factors which influence cellular structure. Cellularorganelles such as ribosomes, mitochondria, etc. are a variety of molecules arranged in aspecific three-dimensional architecture, with the TEM capable of resolution at themolecular level. What is responsible for this cellular ultrastructure that we candistinguish with the TEM? Firstly, is the importance of the most abundant molecule in theliving cell, water, and its effect on other cellular molecules. Water is a charged dipolarmolecule. Being charged, it easily interacts with other charged molecules including thecharged “R” groups of the protein’s amino acids (proteins being the second most abundantmolecule of the cell). Polar water is incapable of interacting with neutral and non-polarmolecules/groups which are designated as hydrophobic (“water fearing”). The chargedgroups are known as hydrophilic (“water loving”). The primary order of protein structureis its amino acid sequence which includes a number of both hydrophobic and hydrophilicunits. Aside from many types of interactions between amino acids of the protein (hydrogenbonding, ionic interactions, disulfide bridges, etc.), the interaction of water with thesehydrophobic and hydrophilic units is crucial to the higher order protein structure,meaning, how it folds into a three-dimensional protein structure. In this scenario,hydrophilic amino acids would be found to the outside of the protein molecule, while thehydrophobic units would be found at the center of the molecule, “hiding” from the external,much more numerous, water molecules. These interactions also explain the orientation ofphospholipid molecules which makeup cellular membranes (phospholipid bilayer).Secondly, we must recognize that cellular physiology and metabolic processes areresponsible for maintaining cellular conditions favorable to the continuity of molecular,

UNIT 1 – PREPARATION OF BIOLOGICAL SAMPLES FOR TEM 7

hence, organelle structure. Examples of some conditions which must be maintained(homeostasis) would include temperature, pH, and osmolarity.

Any changes in these conditions during fixation could lead to structural distortions mainlythrough the process of denaturation. Denaturation, the alteration of the 3-D shape of amolecule (i.e. protein), must be considered when selecting fixatives for TEM specimenpreparation. With the high resolving power of the TEM, any minor alteration in the three-dimensional conformation of molecules is undesirable and would lead to the formation ofartifacts (structures not normally present in the cell which are produced by some externalintervention or agent).You might ask what chemical fixatives are doing and what distinguishes a good fixativefrom a poor fixative, relative to TEM preparation of biological samples. The mainrequirements of a fixative are that it stabilizes cellular ultrastructure without inducingdistortions/artifacts. In stabilizing cellular ultrastructure, the fixative must prevent anundesirable process known as autolysis (meaning self-dissolution/self-digestion) fromoccurring. When an organism dies, cellular lysosomes begin to burst open, releasing aflood of hydrolytic, digestive enzymes into the cell. These autolytic enzymes wouldobviously degrade the very cellular microanatomy which the TEM has the power toresolve. Since autolytic changes proceed rapidly after death, it becomes important tointroduce the fixative to the tissue as soon after death as is possible, ideally within 30minutes.

In summary, fixation must kill and simultaneously stabilize cellular components throughthe prevention of autolysis upon death of the organism (whether it be unicellular ormulticellular). As noted earlier, it is critical that the fixatives do not denature cellularmolecules as a result of their stabilizing components and preventing autolysis.

There are a variety of fixation methods that are utilized world-wide in order to kill andpreserve living materials. Most of these methods are not suitable to the fixation of samplesfor TEM due to the structural distortions/denaturation which result. A list of thesemethods is presented below with an explanation of their value or uselessness to TEMfixation. It should be pointed out that each method is capable of halting autolysisassociated with the death of the organism. This fact qualifies each as a preservative.

• Air DryingThis method is employed for the preservation of harvested grains (wheat, etc.).Drying eliminates the water necessary for autolytic enzyme shape, and therefore,activity. Since all cellular proteins are denatured, this method is poor for TEMfixation.

• PicklingThe pickling process is used to preserve cucumbers (pickles), beets, etc. Preservationis made possible through a change in pH which denatures autolytic and othercellular proteins. The pH is usually lowered (acidic) through the introduction ofacetic acid (vinegar). Due to denaturation, this method is also poor for TEM


fixation.

• AlcoholsAlcohols, such as ethanol (CH3CH2OH) generally make good fixatives since they areall dehydrating agents. Removal of water denatures autolytic enzymes along withother cellular proteins making alcohols unsuitable for TEM fixation if used alone. InTEM (and SEM) fixation, dehydration is an important step in the protocol, however,this process is carried out only after the tissues have already been stabilized withthe primary and secondary fixing agents.

• HeatThe addition of heat energy causes the breakage of numerous bonds which areresponsible for the 3-D conformation of cellular proteins, including the autolyticenzymes. The enzymes are denatured and the organism is preserved. Heat is usedin the canning process. An example would include the cooking of vegetables prior tovacuum packing in the cans. Due to the resulting denaturation caused by heat,TEM samples cannot be simply “cooked” in order to preserve cellular ultrastructure.

• Oxidizers/PrecipitantsPowerful oxidizing agents such as Osmium Tetroxide (OsO4) and PotassiumPermanganate (KMnO4) were the first solitary fixatives used for TEM biologicalsample preparation. Both react at the double bonds present within unsaturatedlipids, such as those found in abundance in the composition of biologicalphospholipid membranes. In addition, and as a result of their reactionwith unsaturated lipids, they introduce an electron dense, heavy metal (Os/Mnprecipitate) to those redox reaction sites. This resultant staining enhances contrastof TEM samples. It should be noted that OsO4 was the primary and only TEMfixative prior to 1963. Since that time it has been discovered that these violentoxidizers can destroy delicate, labile (changeable) cellular structures such asmicrotubules. In 1963 a different primary fixative was proposed for TEM sampleswhich eliminated such redox reaction damage, of especially, proteinaceouscomponents. KMnO4 is such a powerful oxidizer that all of the internal cellularcytoplasmic components are destroyed, with the exception of the membranesystems. If your goal is the study of a particular membrane system, you might wishto try KMnO4 in conjunction with other protocols. Currently, OsO4 is used as asecondary or postfixative due to its ability to react with and stain membranes ofthe cell. It may also react with and stain some protein elements of the cell whichhave been previously stabilized using aldehydes.

CAUTION: Osmium tetroxide is a highly reactive and potent fixative. Thevapors alone can fix exposed epithelial surfaces of the mouth,nose, and especially, eyes (cornea) resulting in temporaryblindness. Crystalline or in aqueous or buffer solution, osmiumtetroxide should be handled with extreme care. Gas tightgoggles, double gloves and a fume hood capable of 150 cfm flow


rate are the minimum safety recommendations.Polyunsaturated oil, such as corn oil, should be at hand in thecase of a spill. It has been advised that approximately twice thevolume of oil be added to an osmium tetroxide spill in order toneutralize it. If spilled in the open, the area should be vacatedimmediately and the proper authorities notified. No oneshould be allowed to reenter without a proper respirator.Waste osmium tetroxide should not be put down the sink butrather, stored in a clearly labeled waste bottle for laterdisposal by environmental carting firms.

• AldehydesAldehydes, containing the reactive carbonyl group (C=O), have been used for yearsin the preservation of biological specimens (embalming, light level histology, etc.). Itis somewhat surprising that they were not considered for TEM fixation prior to1963 when Sabatini, Bensch and Barrnett introduced them as the primary fixativein TEM sample preparation. The role of aldehydes in fixation is their stabilizationof the cellular protein matrix into a somewhat gelatinous state. The carbonyl groupsreact with any reactive amino acid “R” group which in turn results in themethylation (-CH3) of the protein molecules. Neutral methyl groups interact between adjacent protein molecules resulting in velcro-like linkages andstabilization. Since the aldehydes do not denature the proteins, their active sitesremain intact and histochemical and immunocytochemical (ICC) localization ofproteins/enzymes can be performed. The fact that they do not denature make themideal primary fixatives for TEM biological samples. The most common types ofaldehydes used in TEM fixation are formaldehyde (HCHO), acrolein/acrylicaldehyde (CH2 • CHCHO) and the dialdehyde known as glutaraldehyde(CHO-(CH2)3-CHO). While both formaldehyde and acrolein are smaller moleculeswhich diffuse more rapidly into tissue blocks, glutaraldehyde, the dialdehyde, isdoubly reactive and allows for increased stability of the cellular protein matrixthrough the cross-linking of protein molecules. Although glutaraldehyde is the mostcommonly used single aldehyde fixative, an examination of the literature oftenreveals the use of a combination aldehyde primary fixative. In this case,glutaraldehyde is usually used in combination with one or both of the otheraldehydes mentioned above. Conventional formalin solutions are not suitable forTEM fixation. Since formaldehyde is a gas in nature, it must be prepared as anaqueous solution. Through reaction of formaldehyde with water, formic acidformation results. This lowers the pH and serves to denature proteins. In addition,formalin contains methanol which is a dehydrating agent and also denatures.Paraformaldehyde, a purified crystalline trimer of formaldehyde, is the compound ofchoice for TEM fixation. Under low heat for 30min duration, paraformaldehyde goesinto aqueous solution without formic acid formation and without the addition ofmethanol. Although acrolein is an excellent primary aldehyde fixative, it is difficultto work with since it is highly explosive and a potent lachrymator (tear gas).


CAUTION: As fixatives, all aldehydes should be handled with gloves,goggles and under a fume hood, especially acrolein since it is alachrymator. Acrolein is also highly explosive and should bekept away from direct light, heat and flames. All usedaldehydes should be stored in clearly labeled organic wastebottles for later safe disposal. Never put toxins down thedrain!!

✥ Additives

In the literature, you will notice that most fixatives are carried in a vehicle known as abuffer and may also include other additives such as salts (CaCl2) or even sucrose. Themain purpose of these additives is two-fold. Firstly, the buffer is important to themaintenance of the natural physiological pH of the tissue being fixed. Buffer solutions canreact with and counteract the release of excess H+ and/or OH- ions from the tissue beingfixed. As discussed earlier, a change in pH would denature and is therefore not desired.Many types of buffers have been used in TEM fixation including veronal-acetate, PIPES,chromate, phosphate and cacodylate. Even though it has a short shelf life due to theeventual growth of bacteria, phosphate buffers are a good choice since they are non-toxicand therefore, easy to work with out in the open. Once prepared (there are many recipessuch as Sorensen’s), phosphate buffers should be refrigerated at 4˚C to reduce bacterialgrowth. Cacodylate buffer should be handled with care under a fume hood since it containsarsenic. Fixatives are typically made up in the buffer solutions just prior to use. Buffersshould be adjusted to the physiological pH levels of the organism’s internal (or external -as in the case of unicellular protozoa) environment. Mammals and a number of otheranimals range between pH values of 7.2-7.5 with botanical samples approximately 6.8.

The use of additional salts and/or inert substances such as sucrose are for osmolarity/tonicity considerations. The fixative should be isotonic with the internal/external fluidenvironment of the tissue under study. Although trial and error is a common practice indetermining the proper osmolarity, reference sources that list the osmolarity of specificanimal blood/body interstitial fluids are available. Some labs also have access to anosmometer in order to determine precise tonicity requirements for the fixative vehicle.Another additive involves the use of dimethyl sulfoxide (DMSO) which is a penetrantused to enhance infiltration of fixatives into the tissue block.

✥ Fixation Times & Temperatures

In the EM literature, you will find a wide range of fixation times and temperatures used.Who is correct? Can they all be right? Perhaps. One must consider the quality of the finalmicrograph that is being produced. Relative to the duration of each step in a TEM fixationprotocol, there are optimum times of exposure and times which work around technicianschedules for the sake of convenience. In the specific fixation of soft mammalian tissueswhich follows, the indicated times are optimal for ideal preservation with the minimum of


tissue component extraction and artifact production. In the literature you may seeprimary fixation in glutaraldehyde for 1hr to overnight. Under ideal circumstances,fixation through the tissue block will occur within 1hr, provided it is small enough indimension (0.5 cu.mm.). Optimal times should be used whenever possible and practical.

There are two schools of thought on the proper temperature of fixation. One suggests thatfixation should be conducted in the cold, on ice, at 4˚C to slow autolytic enzyme activityand reduce extraction of cellular components. The other suggests that room temperaturefixation hastens fixative infiltration and the actual biochemical process of fixation. In thecold, we are actually slowing the desired process and in some cases, preventing it (if fixtime is inadequate) which can lead to the production of artifacts. The bestrecommendation is to begin the process/protocol at 4˚C and allow it to come to roomtemperature (probably by the time you reach the ethanol dehydration series).

✥ Preparation of Fixatives

• Phosphate Buffers: Used as a vehicle to carry the various fixatives and as a wash. Thesolution is prepared using monosodium (NaH2PO4) and disodium (Na2HPO4) phosphate.Solutions are usually prepared with molarities that are consistent with the tonicity of thesample, such as 0.02M, 0.05M, 0.1M, 0.2M, 0.5M. The molecular weight in grams shouldbe added to one liter of distilled water to yield a 1.0M solution. Simple calculations can beperformed to reduce the molarity from 1.0M and to prepare 100ml of solution vs. 1,000ml(divide by 10). Take note of whether the sodium phosphate is hydrated since the additionof one or more water molecules will increase the gram molecular weight. By way ofexample, to prepare a 1.0M solution of monohydrated monosodium phosphate (NaH2PO4 •H2O), the gram molecular weight, 138g, is added to 1,000ml of distilled water. A 0.1Msolution would be prepared by adding only 13.8g to 1,000ml of DH2O. To prepare 100ml ofthe 0.1M solution, only 1.38g would be added to 100ml of DH2O. In order to adjust the pHof the buffer solution, the proportions of monosodium relative to disodium phosphate mustbe varied according to the chart below.

pH 6.0 6.2 6.4 6.8 7.0 7.2 7.4 7.6 7.8 8.0

NaH2PO4 (in ml) 87.7 81.6 73.5 51.0 39.0 28.0 19.0 13.0 8.5 5.3

Na2HPO4 (in ml) 12.3 18.4 26.5 49.0 61.0 72.0 81.0 87.0 91.5 94.7

Phosphate buffers should be freshly prepared and stored in the refrigerator at 4˚C toreduce the growth of bacteria. The pH should be checked with a pH meter and adjusted asnecessary.

Buffer solution(s) should obviously be prepared first, in advance of any fixative since thefixatives are mixed with the buffer.


• Glutaraldehyde: This primary fixative is available in bottles in aqueous solutions of25% and 50% and in ampoules sealed under nitrogen gas in 8% aqueous solution. Due topolymerization of the glutaraldehyde in high concentrations, the 8% sealed ampoulesafford increased shelf life. Whatever concentration is used, it must be added to anappropriate amount of buffer solution to yield the final concentration (approximately 3% isideal for most purposes). For example, to prepare 3.2% glutaraldehyde in buffer, 20ml of8% glutaraldehyde is added to 30ml of phosphate buffer. The buffered glutaraldehydeshould be made just before use and stored in the refrigerator in the dark.

• Osmium Tetroxide: OsO4 is available as crystals in 0.5g and 1.0g quantities, in sealedampoules, or in aqueous solution (such as 2% and 4%) in sealed ampoules. CrystallineOsO4 is ideal for making larger quantities (50-100ml) of the working solution which isusually set at 1-2%. A meticulously cleaned and dry ground glass stopper bottle with ateflon seal should be used to prepare the working solution. Since crystals of OsO4 are largeand dissolve slowly, it is recommended that the ampoules be held under running hot waterwhich allows the crystals to melt. The ampoule is then gently rolled between the glovedhands to permit recrystallization of the OsO4 in a thin layer inside the ampoule. Theampoule is inserted into the ground glass bottle and the stopper introduced. The bottle isshaken which causes the ampoule to break inside the bottle (Note: EM supply companiescurrently use pre-scored ampoules, some in conjunction with a plastic external cylinder toprevent a cutting injury. The ampoule should be opened, placed in the bottle, and theampoule filled with buffer using a pipette. The bottle is then filled with the remainingbuffer. It is important that the ampoule sink to the bottom of the bottle in order for theOsO4 to go into solution). Once the ampoule is broken, the appropriate amount of buffer isadded to yield the final working solution. To prepare a 1% OsO4 solution, use 1.0g ofcrystalline OsO4 in 100ml of buffer (or 0.5g in 50ml of buffer). When using the solution, becareful not to pipette near the bottom of the bottle since you may pick up some glassshards and introduce them to your tissue samples causing physical/mechanical damage.Once again, it must be cautioned that osmium tetroxide is extremely toxic and should behandled under the fume hood wearing gas tight goggles and double gloves. Cooking oil/corn oil should also be available for possible spills. As with all fixatives, OsO4 is preparedjust before use. It is ideal to prepare the working solution one day before use and leave itout at room temperature overnight. After use, the solution should be stored in therefrigerator. It should be tightly stoppered and placed inside another container of glass ormetal to prevent escape of vapors which will also react with and blacken the inside of therefrigerator.

• Ethanol Dehydration Series: For TEM specimen dehydration, 70%, 95% and 100%ethanol are required. Ethanol concentrations of 95% and 100% are available. It is ideal tomaintain the volume of absolute (100%) ethanol since air in the storage vessel containswater vapor which mixes with the ethanol and reduces the concentration. Small pintbottles are available for one time use since it is imperative that no water remain in thetissues. Another storage technique involves standing the 100% ethanol over a layer ofanhydrous cupric sulfate which serves to absorb water. It should be changed when itbegins to turn blue which indicates that it is hydrated. Do not mix up the cupric sulfate


and EtOH prior to use. The cupric sulfate must be allowed to settle on the bottom of thecontainer.

In order to prepare ethanol dilutions of less than 95%, one should not use the moreexpensive 100% ethanol. In this case, 95% ethanol is used as follows:1. The amount of 95% ethanol in ml, equal to the desired final concentration, is measuredout (for example, a 30% EtOH solution begins by measuring out 30ml of 95% EtOH).2. Distilled water is added to make up a total of 95ml of solution (to complete the above30% EtOH solution, 65ml of DH2O is added to the 30ml of 95% EtOH - total volume is95ml).Ethanol solutions can be refrigerated in the dark until ready for use.

• Propylene Oxide: Propylene oxide is available from EM supply companies usually in250ml quantities. It should be used undiluted. The addition of water would defeat thepurpose of the prior ethanol dehydration series and prevent the infiltration of epoxy resininto the tissues. It is important to know that propylene oxide is extremely volatile. If youare not careful in the solution exchange process, your tissues will dry down leading toartifacts.

Propylene oxide (1,2 epoxy propane) is a small, rapidly diffusing, molecule whichintroduces the epoxy monomer into the tissues thus aiding infiltration. It serves as atransitional solvent as it is miscible with both ethanol and the mixed, unpolymerizedepoxy resin.

CAUTION: Propylene oxide is extremely flammable and should be storedin a cool dark environment. Never expose it to direct heat orflame. It should be handled with gloves, goggles and under afume hood since it is a suspected carcinogen.

• Epoxy Resins: The use of epoxy resins is ideal for the embedment of biological samplesfor ultrathin sectioning for the TEM. Due to its three-dimensional polymerization, samplesare provided with excellent mechanical strength capable of tolerating the forces ofsectioning. The preparation of epoxy resins involves mixing the epoxy compound(s) such as“Epon” 812 and/or Araldite 6005, with specific curing agents including acid anhydrides(DDSA) and tertiary amines (DMP-30). Additionally, depending on the epoxy resin used, aplasticizer such as dibutyl phthalate (DBP) may be necessary. It is critical that allcomponents be thoroughly mixed. The Epon-Araldite mixture used below has provenexcellent for the embedment of soft mammalian tissue samples. The stock solution and thefinal working solution should be hand mixed for at least 15 minutes each. The stocksolution can be frozen for future use. A plastic syringe can be used to store the stocksolutions in exact volumes and with minimal trapped air which could lead to condensedwater vapor contamination of the stock solution as it is allowed to come to roomtemperature. Stock solutions must be allowed to come to room temperature prior to theiruse in the preparation of the working solution.


Mixing of epoxy resins introduce tremendous amounts of air which should ideally beremoved. Air molecules present in the resin mixture would prevent proper infiltration ofthe resin into the tissues. Due to the viscosity of the mixed resin, outgassing throughstanding at atmospheric pressure would be a lengthy and incomplete process. Therefore,mixed resin should be put under a low vacuum environment in order to remove air. Careshould be used in the rate of achievement of vacuum since the resin mixture will overflowthe container, carried out by air bubbles, and contaminate the bell jar. The vacuum shouldbe achieved slowly by regulating the air inlet valve.

Contamination of the work area with unpolymerized resin should be a definite concern. Becareful not to touch doorknobs, refrigerator handles, etc. with contaminated gloves. Neverpour unpolymerized resins into the sink. Resins should be mixed in disposable plasticbeakers using glass rods. Excess resins should be polymerized which renders them safe fordisposal.

CAUTION: Epoxy resins and curing agents can cause contact dermatitisand may be carcinogenic. All resin components should behandled in a fume hood and with double gloves (immediatelydiscard the outer pair if they become contaminated). Whenoutgassing mixed resins, the low vacuum setup should be putin a fume hood. Ovens for polymerization of resins should bevented to the outside or placed in a fume hood.

Although the Epon-Araldite mixture below is prepared volumetrically, the most accurateway to measure it is by weight. Due to the viscosity of the resins, a quantity is certain toremain behind in the serological pipette used to measure it.

Epon-Araldite Mixture

Stock Solution (can be frozen) Small Volume Large Volume

Araldite 6005 12.5ml 25mlPoly/Bed 812 15.5ml 31mlDibutyl phthalate 2ml 4ml

Final Working Solution

Stock Solution 4ml 8mlDDSA 10ml 20mlDMP-30 14 drops* 28 drops*

(*Drops introduced with a Pasteur Pipette)

Another useful formulation is an Epon mixture ideal for embedding directly in plastic orglass containers such as tissue culture plates, petri dishes, microfuge tubes, etc. This


mixture introduces nadic methyl anhydride (NMA) in addition to DDSA. Note the slightdifference in the volume of NMA used in conjunction with original Epon 812 resin vs. the“812” resins available today from EM supply houses (Poly/Bed 812-Polysciences, Inc., EMBed 812-EMS, Inc., etc.)

Epon for Glass/Container Work

Component Volume using Volume using Epon 812 “812” substitute

Epon 812 or 812 substitute 12.5ml same

DDSA 6.5ml same

NMA 6.25ml 9.0ml

DMP-30 1.0ml same

DBP 0.25ml same

Mixed resins will polymerize at 60˚C for 48 hours. If heat is a concern, epoxy resins willalso cure under ultra-violet (UV) light.

✥ Methods of Fixation

• Immersion: In this type of fixation, the tissue of interest is removed/excised from theorganism (or if small enough, the entire organism) and placed into the primary bufferedfixative such as glutaraldehyde. The buffered primary fixative can be put into petri dishesor as large drops on a card of dental wax. When a complex animal (mammal, etc.) is used,it must be sacrificed quickly, followed by opening the body cavity, identifying the organ(s)/tissue(s) of interest, and rapidly excising them using a razor blade or scalpel. Once thetissues are transferred to the fixative, they must be minced to blocks which are less than1.0mm3 in dimension (0.5mm3 is optimal) using clean single-edged razor blades. Mincingshould be done with care since mishandling will result in mechanical damage artifacts.Artifacts will also arise if the tissue pieces are allowed to dry. It is imperative that they bekept submerged in the fixative solution at all times. Once minced, the tissues aretransferred to small vials containing the primary fixative and allowed to remain for theallotted time (usually about one hour). All subsequent steps in the protocol (with theexception of epoxy resin embedding) can be carried out in these vials through decantingthe current solution and quickly, yet carefully, introducing the next solution in theprotocol.


• In-situ: In-situ fixation, meaning ‘in the original place’, is accomplished by bringing/applying the primary fixative to the specimen of interest. By example, fixation of a thinplant leaf is conducted by placing a ring of petroleum jelly on the leaf surface and fillingthe ring with the primary fixative. Once fixed, the area of interest is cut out, minced andplaced into vials to continue with the protocol. Another example involve the fixation oftissue culture cells. The fixatives are simply administered to the culture dishes throughpouring them on and off of the cellular monolayer. In the animal fixation described aboveunder immersion fixation, once the animal is humanely sacrificed and the body cavity cutopen, primary fixative should be poured into the cavity in order to initiate fixation.

• Vascular Perfusion: This technique uses the vascular system of an animal to deliverthe primary fixative deep into the tissues and directly on target. This is probably the mostoptimal means for administering the fixative. Delicate tissues subject to rapid post-mortem change must be fixed in this manner. A good example would be nervous tissues. Inthis procedure, the animal is anesthetized and the heart and major blood vessels exposed.Typically, a canula is inserted into the aorta and a balanced saline solution is allowed togravity feed into the blood vessel. Eventually, the saline solution is cut off by a clamp andthe fixative is allowed to flow into the vascular system. The tissues of interest can be laterexcised, minced, and placed into vials in order to continue the process.

• Fixation by Vapors: Small delicate surfaces, such as membranes, may be fixed in thismanner. The specimens are simply suspended over a solution of osmium tetroxide, usuallyovernight. The samples will blacken to indicate that fixation has occurred. Samples can bedehydrated and embedded in resin for ultrathin sectioning.

In our lab, we use a combination of in-situ and immersion fixation of biological samples asdescribed above. Once again, care and common sense should be used in thehandling of all fixatives!

✥ “Routine” Biological Soft Tissue Protocol

In fact, no single “routine” protocol for the fixation of soft biological tissues exists. Thereare as many protocols as there are investigators, with no one better than the other. Aslong as your protocol enables you to observe the cellular features that you are interestedin, it is valid. Of course, protocols will vary based on the particular organism beingstudied. It is apparent that botanical samples should be fixed using different protocolsthan animal samples, however, protocols will vary even among related organisms. By wayof example, metazoans of different classes, orders and even genera will have variedphysiologies which give rise to a wide range of body/interstitial fluid characteristics. Asdiscussed earlier, in order to prevent artifacts, the buffer pH and tonicity would have to beconsidered for each animal investigated. In addition, conditions of fixation should beconsidered. Are you fixing under ideal laboratory conditions or are you in a tropical fieldsetting? How much time will you have to mince the tissues after excision? You may haveto store tissues in tropical climates for days to weeks without the ability to precisely mincethe tissue blocks until you return to the lab. Even under these unfavorable conditions, you


can modify your fixation protocol to yield quality results. In a study of neo-tropical bats,Nagato, Tandler and Phillips used a trialdehyde-DMSO primary fixative which consistedof 1% glutaraldehyde, 1% paraformaldehyde, 0.5% acrolein, 2.5% dimethyl sulfoxide, and 1mM CaCl2 in 0.05 M cacodylate buffer (pH 7.2). The tissue was stored for 14 days intropical conditions before being transferred to 3% glutaraldehyde at 4˚C. Given thecircumstances, the resulting TEM micrographs were of extremely good quality.

The protocol which shall now be outlined relates primarily to soft mammalian tissuesamples prepared under ideal laboratory conditions. The solutions are initially at 4˚C andare allowed to come to room temperature. The steps in this “routine” protocol are asfollows:

• Primary Aldehyde Fixation - used singly, buffered glutaraldehyde (usually 3-4%) isthe most common choice for TEM fixation of biological tissues. Using methods of fixationdescribed earlier, the primary fixative is delivered to the tissue of interest. In this lab, themost common method employed for soft mammalian tissues is in-situ followed byimmersion fixation in petri dishes. The tissue is minced carefully using two clean single-edged razor blades into pieces no larger than 3.0 cubic mm, with the ideal tissue block sizeno larger that 1.0 cubic mm. If you wish to complete mincing in the primary fixative, youwill need to reduce the tissue blocks to 0.5 cubic mm, however, this can wait until the finalbuffer wash just prior to osmium tetroxide (OsO4 does not penetrate as well as thealdehydes). Once minced, the tissue blocks are transferred to small glass vials containingthe primary fixative.

The aldehydes will penetrate the tissue block to fix cellular proteins as described earlier.The ideal fix time for aldehydes is approximately 1 hour, however, tissues can be stored inglutaraldehyde at 4˚C for a number of days. I recall having stored samples inglutaraldehyde for as long as 1 year without the development of major structural artifacts.

• Buffer Wash - usually the same buffer is used as is used to prepare the aldehydefixative. In this lab, phosphate buffers are used. The buffer wash is done in order to washout any unbound aldehydes which would react with the OsO4 in the next step, leading to acontaminating precipitate reaction. This wash is usually done a number of times (3 times)for ten minute intervals.

• Osmium Tetroxide Postfix - buffered OsO4 in 1-2% solutions is administered to thetissues next. At this point, the tissue blocks must be on the order of 0.5 cubic mm indimension. The OsO4 primarily reacts with unsaturated fats in the cell and imparts anelectron dense heavy metal staining to regions of the cell which are composed of theselipids (such as cell membranes). OsO4 also reacts with some cellular proteins, however,because aldehydes were used first, the proteins should now be stable and not subject todenaturation by this powerful oxidizing agent.

OsO4 postfixation is usually carried out for 1 hour with the tissue samples blackening toindicate the activity of the fixative.


• Distilled Water or Buffer Wash (Optional) - many investigators use a brief wash ofeither distilled water or buffer to remove excess OsO4. The wash is performed since theOsO4 may react with the ethanol in the following step. At times, a cloudiness may developin the fixation vials if this step is avoided. In this lab, we usually dispense with this washsince the cloudy result is rare and short-lived, usually lasting for the initial ethanolchange. If this step is performed, usually 2-3 changes of ten minutes each is sufficient.

• Dehydration Series (Graded Ethanol/Acetone Series) - the tissues are passedthrough an ascending dehydration series of either ethanol or EM grade acetone. Toprevent distortion and shrinkage, many individuals ascend slowly to 100% ethanol oracetone, starting at 10% and ascending in 10% steps (10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%). In order to preserve delicate, internal labile structures, it is alsoimportant to get the tissue out of an aqueous environment as soon as possible, therefore,we start dehydration of samples for TEM at 70% (two changes for ten minutes each). Wethen proceed to 95% (two changes for ten minutes each) and then to 100% (two changes forten minutes each), typically using ethanol. The 100% ethanol vials must be completelyfilled to prevent air (containing water vapor) from reducing the concentration. Theabsolute ethanol should be standing over anhydrous cupric sulfate before use to ensure itis not hydrated. This is a critical step since any water remaining in the tissue will preventepoxy resin infiltration. The dehydration series is performed since water and mostembedding media (epoxy resins) are not miscible.

• Transitional/Intermediate Solvent (Propylene Oxide) - this is an optional step inthe protocol which will ensure successful epoxy resin infiltration. Propylene oxide (1,2epoxy propane) is a highly volatile, flammable, small molecule which rapidly penetratesthe tissue blocks. It serves to enhance infiltration of the tissues by epoxy resin since itcarries in the reactive epoxy monomer. If used, three changes for ten minutes each isadequate.

• Propylene Oxide : Mixed Epoxy Resin (1 : 1 ratio) - in this step, an equal volume ofpropylene oxide and mixed (unpolymerized) epoxy resin is introduced to the tissues. Thepurpose is to make a gradual transition to pure epoxy resin and enhance its infiltrationinto the tissue blocks. This step is carried out in vials for 1 hour.

• Vacuum Infiltration - after the pure mixed epoxy resin has been placed under a lowvacuum (bell jar and rotary pump setup) to eliminate air introduced through the mixingprocess, a small volume is poured into a small diameter petri dish (enough to fill thebottom and produce a thin uniform layer). Tissue blocks are selected and placed into theresin using toothpicks or bamboo sticks. The labelled petri dishes are placed into the lowvacuum setup and the bell jar is pumped out. This process is carried out for 1 hour toprevent gas from impeding epoxy resin infiltration.

• Embedding - During the 1 hour vacuum infiltration process, BEEM (Better Equipmentfor Electron Microscopy) capsules (which have a preformed truncated pyramid tip of 1.0square mm) are stuffed with an identification label and filled with epoxy resin mixture to


a slight negative meniscus. Allow air bubbles to break naturally and further fill capsulesas needed.

Identification labels should identify the investigator and the tissue type. Write the labelsin pencil only since pen and other inks can react with the resins. Once written, the labelsare wrapped around a wooden stick (bamboo) and placed into the BEEM capsule. Don’twrap the label too tightly around your stick since you want the label to spring against theinner walls of the capsule. When viewed from above, the labels should be barely visible. Ifthey project into the center of the BEEM capsule, try to push them against the sides withtwo sticks. You do not wish the labels to impede the descent of the tissue blocks to the tipof the capsule.

With the BEEM capsules labelled and filled with the epoxy resin mixture, individualtissue blocks can be transferred from the petri dishes to the center of the filled BEEMcapsules using a pointed bamboo stick. The tissue blocks are heavy as a result of osmiumimpregnation and will sink to the capsule tips prior to the polymerization of the resin.

BEEM capsules should be supported in some type of holder. In this lab we use oldmicropipette tip holders. Some EM supply companies manufacture BEEM capsule holdersand some individuals simply punch appropriate sized holes into cardboard squares.

With the tissues transferred to the BEEM capsules, the holders are placed into an oven at60˚C for at least 48 hours in order that the epoxy resin polymerize. All containers (plasticbeakers, petri dishes, etc.) which came in contact with the epoxy resin should also be putin the oven and be allowed to polymerize for safe disposal. Vials which held the 1:1propylene oxide/resin mixture should be left open under a fume hood for a number ofhours to overnight to allow for the evaporation of the propylene oxide (never putpropylene oxide near a heat source/oven). The vials can later be placed in the ovensto polymerize the epoxy resin and finally discarded.

After 48 hours, the BEEM capsules can be removed from the ovens and stored at roomtemperature. The tissues are ready for trimming and ultrathin sectioning.

✥ Tissue Processing Note

From the mincing of tissues in the primary fixative and up to the point of vacuuminfiltration, tissue blocks are handled and contained in small glass vials with plastic snap-caps (8ml capacity). Chemical agents are simply decanted using a Pasteur pipette and thereplacement agent added with a different pipette. Do not allow your tissues to drydown during processing. This will lead to denaturation and artifacts. Whendecanting, always leave a small amount of the original solution to cover the tissue blocks,especially when decanting the highly volatile ethanol (especially 100%), acetone andpropylene oxide.


✥ Embedding Media

In order for electrons to pass through a sample in the TEM, it must be reduced inthickness to between 600-900Å. Therefore, the sample must be sectioned ultrathin.Ultrathin sectioning would be impossible unless the biological soft tissues we study arestrengthened to withstand the incredible forces encountered during sectioning. Unless asuitable embedding media is used, the soft tissues would simply disintegrate at thecutting edge along with the fine structures we wish to see.

An adequate embedding media for TEM samples must meet certain requirements whichfollow:

1. consistency - a formulation must yield the same results for each mix.2. availability - components must be readily available.3. purity - components must be identified and characterized to avoid artifacts.4. solubility - in common solvents.5. miscibility - with other embedding media/curing agents/dehydrating agents (e.g.

alcohol).6. viscosity - low is ideal from a standpoint of convenience.7. polymerization - must be controllable, uniform, and occur in a reasonable amount

of time.8. transparent - to light (to view tissue in blocks and sections) and electrons.9. stability - under the electron beam.10. sublimation - avoids liquid interface and resultant surface tension forces.11. cross-linked polymer - for good ultrastructural preservation / strengthens

tissues.12. stainability - allows for heavy metal (TEM), pigment (LM) and histochemical

staining.13. no shrinkage - little volume change during polymerization.14. stores well - long shelf life (some preliminary mixtures can be stored in the

freezer).

In general, resin components should be mixed thoroughly to avoid uneven polymerizationand embedment. Hand mixing with a clean glass rod for 15-20 minutes is usuallysufficient. Of course, this mixing will introduce a great deal of air to the mixture. Since themixture is viscous and lengthy staining times may cause separation of the components, alow vacuum (bell jar / rotary pump) setup can be used to eliminate gas from the mixture.Such a setup is best run under a fume hood to avoid resin fumes.

Another important consideration is that the hardness of the resin should closely match thehardness of the tissue in question. This will be considered in the discussions of the variousembedding media which follows.


• MethacrylatesThe first embedding media to provide good results for the embedment and sectioning ofTEM samples were the methacrylates which were introduced by Newman, et al. in 1949.These resins are low in viscosity and their hardness can be adjusted by varyingproportions of methyl versus butyl methacrylate. The catalyst typically used to polymerizethe methacrylates is benzoyl peroxide. Initially, they were hailed as the ideal TEMembedding agent until it was demonstrated that they polymerize unevenly. Since they arelinear polymers, it was noted that they shrink nearly 20% during the polymerizationprocess. This leads to a flowing of cellular components and gross distortions known as“explosion artifacts” due to the presence of large, seemingly vacuolated regions. Inaddition, the methacrylates are very unstable under the electron beam. This clearing ofthe resin produces an increase in contrast (since only tissue components are left behind).However, due to surface tension which arises at the liquid resin/tissue interface and theflowing and subsequent collapse of cellular structures, distortion artifacts are a certainty.Formvar (plastic) coated grids were typically used to support methacrylate sections.

• Epoxy ResinsIntroduced in Denmark for TEM by Maaløe and Birch-Anderson (1956) and later inEngland by Glauert, et al. (1956), the epoxy resins are and continue to be the bestembedding media for biological samples. The first quality epoxy resin developed for use inTEM sample embedments was “Araldite”, an extremely viscous compound which was usedfor many years in England by model makers. Later, the epoxy resin known as “Epon” 812was developed and eventually marketed by the Shell Oil company. Today, many “812”epoxy resins are available from EM supply companies (PolyBed 812-Polysciences, Inc., EMBed 812-EMS, Inc., etc.). As discussed earlier, in this lab, we use an Epon-Aralditemixture which is suitable for a variety of tissues.

Epoxy resins are transparent, yellowish, cross-linked, thermosetting (with the applicationof heat) polymers which are highly viscous in their unpolymerized form. The modern epoxyis a diepoxy which has an epoxy group at each end of the molecule. The epoxy group is astrained, three-membered ring (C-O-C) which when ruptured, provides the energy 22Kcal/mol) to drive the polymerization forward. Spaced along the organic molecular chainbetween the two epoxy groups are 1-5 hydroxyl (-OH) groups. Both the epoxy groups andthe hydroxyl groups react with a variety of “curing agents” and either heat or ultra-violet(UV) light to yield the three-dimensional, cross-linked polymer. The final mechanicalproperties of the embedment are based on the type of epoxy resin and curing agents used.

The epoxy group reacts with any reactive hydrogen atom which leads to its rupture andsubsequent release of energy to drive the polymerization. A good source of reactivehydrogen atoms is the organic molecule class known as tertiary amines. The tertiaryamines such as Benzyldimethylamine (BDMA) and Tridimethyl amino methyl phenol(DMP-30) are also known as catalysts or accelerators and are responsible for linear(end-to-end) polymerization. They should be added last and just before use of the epoxymixture. Another class of organic molecules known as acid anhydrides react with thehydroxyl groups of the epoxy resin molecule to provide cross-linking. Acid anhydrides such


as Dodecenyl succinic anhydride (DDSA) and Nadic methyl anhydride (NMA) are alsoknown as hardeners.

The mixture of epoxy resin (Epon/Araldite), acid anhydride hardener (DDSA), tertiaryamine catalyst (DMP-30) and heat (or UV) yields a strong three-dimensional polymerwhich is resistant to solvents and heat. The three-dimensional polymerization providesexcellent mechanical strength for ultrathin sectioning without shrinkage and resultantexplosion artifacts. The ultrathin sections are extremely stable under the electron beam,so much so that uncoated grids can be used to pick up the sections.

As mentioned earlier, it is important to match resin and tissue hardness. Hard tissueembedded in a soft resin would break out of the resin block. Soft tissue in a hard resinwould be distorted or disintegrate as it is moved across the cutting edge. Much of thismatching is a trial-and-error process of working with different resins and curing agents.By example, when using Araldite with the hardener NMA, the resultant polymer is toohard and brittle. When using DDSA, the block is still too hard for biological tissues. In thiscase a modifier such as Dibutyl phthalate (DBP) is used. This compound is nonreactive,however, it increases elasticity to produce a softer embedment. DPB is also known as a“plasticizer”. In another example, when DDSA is used with Epon, the block is too soft.Conversely, when NMA is used with Epon, the block is too hard. This situation is idealsince final hardness can be adjusted by varying the proportions of DDSA and NMA.One should be reminded that most epoxy resins are immiscible with water and thatcomplete dehydration must be carried out prior to the introduction of epoxy. Any water inthe tissue will result in a poor embedment. The transitional solvent, propylene oxide, aidsin the infiltration of epoxy resins into the tissue and its use is recommended whenpossible.

• Other Embedding MediaOther types of embedding media for TEM samples exists. Polyester resins such asVestopal W and Rigolac provide an improvement over methacrylates since they are notsubject to shrinkage due to three-dimensional polymerization. They are polymerized byheat, light and oxygen and should be protected from such sources by keeping mixturesrefrigerated in the dark.

Another useful resin mixture is known as Spurr’s resin (vinylcyclohexene dioxide-VCD)which is a low viscosity resin ideal when infiltration of the sample may be difficult.Botanical samples are often embedded with Spurr’s resin since the cell wall may provide abarrier to the penetration of high viscosity resins.

Water soluble resins are available such as Aquon which is prepared by extraction of thewater soluble fraction of Epon. Aquon is completely miscible with water at 15˚C andbelow. Glycol methacrylate (GMA) is also relatively water soluble for samplepreparation where dehydration would be impractical or detrimental to the tissue.


Fixation ScheduleMammalian Soft Tissue Protocol - In-situ and Immersion fixation - 0.5mm3 tissue blocks

Initial Fixation at 4˚C - Ascending to Room Temperature

Schedule Duration

3% Glutaraldehyde (0.2 M phosphate buffer, pH 7.4) 1 hour

Buffer Wash 3 x 10 minutes each

1% OsO4 (buffered as above) 1 hour

Buffer or DH2O Wash (optional) 2 x 10 minutes each

70% Ethanol 2 x 10 minutes each 95% Ethanol 2 x 10 minutes each 100% Ethanol (fill vials completely) 2 x 10 minutes each

Propylene Oxide 3 x 10 minutes each

1 Propylene Oxide : 1 Mixed Resin 1 hour

Vacuum Infiltration (in small petri dishes) 1 hour

Embed (in pure Epon/Araldite mixture) 48 hours at 60˚C Place labels into BEEM capsules, then add degassed resin, then tissue blocks. Tissue blocks will descend to the tip of the EEM capsules before polymerization of resin.


Fixation Schedule WorksheetMammalian Soft Tissue Protocol - In-situ and Immersion fixation - 0.5mm3 tissue blocks


Schedule Time In Time Out

3% Glutaraldehyde (0.2 M phosphate buffer, pH 7.4)

Buffer Wash

1% OsO4 (buffered as above)

Buffer or DH2O Wash (optional)

70% Ethanol 95% Ethanol 100% Ethanol (fill vials completely)

Propylene Oxide

1 Propylene Oxide : 1 Mixed Resin

Vacuum Infiltration (in small petri dishes)

Embed (in pure Epon/Araldite mixture) Place labels into BEEM capsules, then add degassed resin, then tissue blocks. Tissue blocks will descend to the tip of the BEEM capsules before polymerization of resin.


✥ Final Chemical Fixation Considerations

Throughout the process of chemical fixation, the electron microscopist must be remindedthat this is not a natural event and that the final result must be considered artifact. Allimages of fixed biological samples produced by the TEM are artifactual, not “true-to-life”.Does this mean that the cellular structures which have been previously described truly donot exist? Do double membrane bound structures known as mitochondria, which possessinternal infolded cristae, really exist? Are they simply an artifact of preparation? One cananswer those questions from an examination of the scientific literature. When you see arecurring feature which possesses the identical structural intricacies and occurs in regionsof the cell with specific requirements, in this case energy, we can be confident as to theexistence of this structure. At the same time, one must be extremely careful not to induceartifacts of preparation which can arise from improper handling of the samples. Poortechnique throughout the entire fixation protocol will lead to glaring defects and artifactswhen finally viewed under the high resolution of the TEM.

Finally, while conducting a fixation protocol one must be cautioned that each step mayinvolve your exposure to highly toxic chemical agents. Remember that fixatives kill andpreserve living tissues, including yours! A healthy respect for all potentially hazardouschemicals should be developed by the student of science/biology. Use common sense inworking with all chemicals! Assume that all chemicals you are unfamiliar with arehazardous and use appropriate precautions such as the use of disposable gloves (two pairsif necessary), goggles, and the fume hood. The ideal course of action is to consult theMaterial Safety Data Sheet (MSDS) for the chemicals you will be exposed to and followthe listed safety guidelines. MSDS should be available in any location that involves thehandling of chemicals. Specific precautions will be found both in association with anychemical listed in any fixation protocol in this book. Whether a chemical agent has animmediate effect (contact dermatitis, blindness, death) or a latent and/or cumulative effect(cancer), the goal is to eliminate exposure of both yourself and future students to theagent. Be careful not to touch bare tabletops, door handles and knobs, etc. withcontaminated gloves. You will be contaminating the work area for unsuspecting personnelthat would come into bare-handed contact with these surfaces, possibly for years to come(especially in the case of resin components). Also, be aware of the location of emergencyequipment in the lab, such as fire blankets, fire extinguishers, eye wash and showerstation(s). The phrase “Look before you leap”, meaning, think before you do anything,is most appropriate when dealing with dangerous chemicals in the laboratory.


Chapter 2 - Ultrathin Sectioning & Ultramicrotomy

Unless the electron beam produced by the electron gun of the TEM can pass through thespecimen (or transmit - hence the name Transmission Electron Microscope), achieving animage of high quality and resolution, or any image at all, is impossible. In order for theelectron beam to pass through a sample, it must be sufficiently thin. This level of thinnessrequired has been determined to be between 600-900Å, what is conventionally known asultrathin. Since most samples, biological or otherwise, which one would consider viewingunder the TEM are much thicker than this, a process known as ultrathin sectioning isrequired. It wasn’t until the early 1950’s that quality ultrathin sections were cut routinely.It is for this reason that biological TEM lagged behind the development and commercialavailability of the TEM; the first commercial TEM was marketed by the Siemenscorporation in 1939 ( an instrument designated the Elmiskop I).

The precision instrument which was developed and makes it possible to cut ultrathinsections is the ultramicrotome. The following information covers the theory and use of theultramicrotome along with the related requirements which are necessary for its use,including block trimming and glass knifemaking/diamond knife usage.

✥ Processing of Embedded Blocks - Block Trimming

Prior to ultrathin sectioning, polymerized blocks must be trimmed freehand under adissecting microscope. This process is conducted since tissue blocks rarely descendcompletely to the tip (truncated/flat face) of the BEEM capsule. This results in anembedded tissue block which is somewhere beneath the blockface. When you consider howlittle material is removed from the face through ultrathin sectioning (600-900Å), itobviously becomes necessary to expose the actual tissue sample at the surface of the block,otherwise, you will be sectioning epoxy resin only. Unlike in the preparation andsectioning of samples for the light microscope, TEM sections should be completely filledwith tissue sample. In LM sectioning, a considerable portion of the section periphery is theembedding material, such as paraffin, with the actual tissue centrally located.

Block trimming is a vital step which will ensure that your sections will contain amaximum amount of tissue. After considerable effort is taken to properly fix your tissuesamples, careful block trimming is critical to success at the ultramicrotome.

Block trimming is conducted entirely by hand under a dissecting microscope, preferablywith a bright overhead light source. Since the process is entirely manual, many studentsfind this to be one of the most difficult procedures to learn in TEM specimen preparation.The ideal setup is an older style Bausch & Lomb stereoscope with a “pod” head that is fitwith 15X oculars and yields a maximum magnification of 45X. A bright external B&Lilluminator is used in conjunction with the stereoscope, a setup which is far superior to anewer model B&L stereoscope with its less intense light source focused from overhead by amirror.


Besides the microscope setup described above, block trimming also requires an embeddedblock (in a BEEM capsule), some type of chuck (such as a collet chuck) for holding theblock, a trimming base or stage for supporting the chuck, a pair of pliers for removing theblock from the BEEM capsule, a package of double-edged razor blades, a beaker of acetone(with a cover) to remove oil from the razor blades, Ross lens tissue to dry the acetonedipped blades, and a metric ruler in order to determine the dimensions of the trimmedblockface. It should be noted that although most individuals in the field do not recommendthe use of double-edged blades, they are by far sharper than single-edged razor blades.The minor inconvenience of learning to use these highly flexible blades can be translatedinto much less difficulty at the ultramicrotome. The more rigid single-edged blade is notnearly as sharp and results in trimmed blocks with what we refer to as a “frosted” faceand sides. These frosted areas appear smooth, however, they are actually irregular andcan lead to sectioning problems such as sections which do not completely detach from theblockface as they are cut.

Prior to block trimming, the polymerized block must be removed from the BEEM capsule.A simple way to proceed would be to slit the side of the capsule with a single-edged razorblade. If one is not very careful, this technique often results in numerous cut fingers. Therecommended method involves using a pair of pliers to force the block out of the capsuleusing pressure. The BEEM capsule is first squeezed on its side which results in anoticeable separation of the resin from the capsule’s plastic. The pliers are then positionedat the capsule tip, just above the circular ring, superior to the truncated BEEM pyramid.Slow and even plier pressure is applied which results in the block moving outward fromthe capsule. Do not bring the jaws of the pliers together as you might damage theblockface. Instead, reposition the pliers slightly higher on the BEEM capsule andcontinue. Once this has been done enough, at least one-half of the block will project fromthe BEEM capsule. You should be able to twist and pull the block from the capsule at thispoint where it will be inserted about two-thirds of the way into a chuck. Empty BEEMcapsules can be saved to place over the tip of a trimmed block and protect it until the timeof sectioning.

For the Sorvall-Porter Blum MT-2B ultramicrotome, collet chucks, with circular openingswhich are reduced in diameter upon tightening (as found in the design of a drill) areemployed. It is best to load the blocks horizontally, from the side so that you don’t have tocombat gravity. As stated earlier, blocks should be loaded about two-thirds of the way intothe chuck. The threaded chuck is then screwed into the trimming base which is placedonto the stage of the stereoscope. The blockface is focused on at maximum magnification(45X) and brightest overhead illumination. The 1.0mm square blockface formed by theBEEM capsule is then set square in the field of view by rotating the trimming base.

A double-edged razor blade is placed for a few seconds in a 50ml beaker of acetone. It isremoved and wiped dry by pulling a sheet of Ross lens tissue away from the each edge.The blade should be held with the index fingers of each hand on top of the blade and thethumbs underneath. Fingers of each hand should be in close proximity with a slightpulling against each other to increase blade tension and rigidity. The other three fingers of


each hand are anchored on the trimming base with the block and chuck between. Cuttingshould occur away from the individual, however, some cut the top (across the blockface)towards themselves with success.

Initially, the blockface is cut down to the tissue. Thin sections are cut which are as parallelto the original blockface as possible. The blade should be held level or even better, at avery slight downward angle which ensures that the blade will dig in and produce auniform and smooth new face. A slight blockface angle may result which can becompensated for on the ultramicrotome. A sharp blade results in a characteristic striatedappearance on a reflective blockface. If “frosting” appears, the region of the blade beingused has dulled and should be moved to a sharp area. For routine work, your goal is totrim the face down to the tissue block. You can be sure that the block has been reachedwhen black material appears in your hand trimmed sections. This black material is yourosmium fixed sample. At this point, your blockface is much larger than the original 1.0mmsquare and must be reduced by trimming the sides of the block. Sides should be trimmedparallel to the angle formed by the original BEEM capsule (~45˚) and not very deeply. Theideal pyramid is short and broad-based. Deep, steep sides leads to a poorly supportedpyramid which vibrates when sectioned leading to a problem known as “chatter”.

As the sides are reduced, so is the blockface. The ideal final dimensions of the blockfaceare 0.25mm on the longest side(s); not to exceed 0.5mm, with the longest sides parallel toeach other. If the longest sides are not parallel, the ribbon of sections that come off theknife edge during sectioning will curve back to the edge instead of moving out in a straightline. A number of final blockface shapes can be trimmed such as a square, rectangle, andtrapezoid. The trapezoid is the best shape to trim since it is most probable that sections ofthis shape will form what are known as ribbons. The following illustration (fig. 1) shows ablock before and after trimming.

Fig. 1

1.0mm

1.0mm 1.0mm

0.25mm

0.25mm

Block Trim

Side View

Top View

Tissue

Tissue Tissue

Tissue

} Razor Blade Hand Trim Lines


In closing a discussion of block trimming, it must be noted that most ultramicrotomes canbe used to trim blocks using glass knives since the knife stage and specimen arm anglescan be manipulated through a wide range. Additionally, automatic block trimming devicesand even gadgets to remove blocks from BEEM capsules are marketed. Although suchinstruments exist, for a price, most labs still trim blocks by hand.

✥ Glass Knife Making

A suitable cutting edge for use in ultrathin sectioning is mandatory. Metal edges cannot besharpened adequately to cut sections on the order of 600-900Å. The use of free fracturedglass knives, introduced by Latta and Hartmann, was an important advance to theeventual production of ultrathin sections. The sharpest cutting edge that can be producedis that of free fractured glass. Even our ancestral cave dwellers knew the value of brokenglass as they fashioned obsidian (volcanic glass) into tools for cutting. Today somesurgeons will use obsidian scalpels to reduce “hamburgerization” of the integumentary(skin) tissues which can lead to scars. Only a handful of such courageous surgeons will useobsidian scalpels since they risk cutting their own valuable and skilled hands.

In glass knifemaking (see fig. 2 on opposite page), plate glass strips are used. In this lab,LKB glass strips are used. LKB (now Leica) manufactures fully stress flown glass strips inwhich lines of stress are virtually eliminated. These high quality glass strips produce thesharpest eventual cutting edges. When a piece of glass is free fractured, it is preciselyscored using a carbide scoring wheel or diamond tipped scribe. The score line must not runentirely across the length of glass you are trying to break, from one edge to the other. Onthe contrary, the score must be along the midline of the length of glass to be broken, at ornear the center. Pressure is then applied beneath the score line and the glass freefractures to yield the sharpest cutting edge known. The simplest and least expensive wayto apply this pressure is through the use of glazier’s pliers. The only problem here isinconsistency in the quality of the glass knives. In the early days of electron microscopy,one individual would be hired for the job of making glass knives. This person wouldbecome proficient at glass knifemaking through repetition and could routinely makequality edges for ultrathin sectioning.

For many years, instruments have been available to somewhat automate the process ofglass knifemaking. These instruments, such as the LKB 7800 series knifemakers, allowanyone to manufacture quality glass knives. The LKB knifemaker has provisions forprecisely aligning, clamping, scoring and breaking glass strips of various dimensions. Themost common dimensions of the glass strips purchased to make glass knives are 9-18" longx 1.0" wide x 1/4" deep. Shorter glass strips (9-10") are easier to handle and the LKBknifemaker can be used to cut longer strips in half. To cut a long strip in half, the strip islaid on top of the two spring loaded posts and the end of the strip is lined up even with thesmall black dot mark on the right-hand side of the machine’s surface. The strip is scoredand broken as per the procedure for making a 1.0" square described below.


Fig. 2 – Glass Knifemaking:

1 inch

1 inch

cutting edgeconchoidal fracture plane

base (1 mm)

Boat/Trough Attachment:

tape seal

water level

1 inch

1 inch

score line free break path

1/4 inch

frilling

score line


Before making a free fractured knives, the glass strip must be carefully cleaned in a non-filming soap such as Liquinox. It should be held at one end only, the end which will not beused to make the knives, and thoroughly rinsed by holding the end used to make theknives upright. This prevents any soap on your hand which holds the glass from runningdown the strip and contaminating it. A thorough rinse in tap water followed by a finalrinse in distilled water is recommended. The strip is then rested at an angle against lintfree cloth, with the useful end upright, and allowed to air dry. Don’t wipe the glass dry,even with so-called lint free cloth, as you will produce a static charge on the glass andattract dust. Once dry, you can proceed with glass knifemaking on the LKB 7800.Initially, the LKB knifemaker should be set up with the breaking knob, the large blackknob on the lower right of the machine which is used to apply pressure, fully counter-clockwise. The clamping/scoring head should be elevated in place using the ball-endedclamping lever and the silver scoring adjuster should be on the “lines” setting (the threelines should be facing up - the other positions are 2.5 and 3.8).

Holding the clean glass strip by one end, it is carefully lowered to the surface of the LKB7800 with its scored/frilled edge down. The strip can be touched on the upper 1.0" widesurface but not on the 1/4" deep edge. Touching the strip on its surface, it is pulled downflush with the white plastic guide (which is set at 90˚) and slid to butt up against the firstof two metal posts. The clamping/scoring head is lowered using the clamping lever until itcontacts the glass strip surface. Upon contact of the clamping head, immediately releasethe glass strip surface with your hand. Failure to release with your hand at this point willresult in the scored glass not breaking. Lower the clamping lever so that the ball end isjust above the surface of the machine (finger width space). With the score adjuster set on“lines”, score the strip by smoothly pulling the white scoring lever out. When the scoringlever is pulled, a carbide wheel descends and ascends in an arc to avoid scoring the glassfrom edge to edge. This score is made perpendicular to the length of the strip, exactly 1.0"in from the end. The large black breaking/pressure knob is then turned slowly andconsistently until the glass free breaks. When the glass breaks, the breaking knob isturned back full counter-clockwise and the clamping head is elevated using the clampinglever while supporting the head using the extended scoring lever. At this point, a separate1.0" glass square has been produced. The remaining glass strip can be removed and placedon lint free cloth.

The glass square can be slid to the top of the pair of black pressure feet and carefullyrotated counter-clockwise into a diamond formation, by touching only the top surface ofthe square. The lower edge of the diamond should be slid between the two white plasticsupports with the upper edge aligned across from the inverted “U” of the upper metalholder. The upper holder is brought into place by pushing in and rotating the small blackcontrol knob on the rear left side of the machine. The clamping head is lowered as beforeand the scoring adjuster is set to 2.5. Before scoring, the small black damper is moved tojust touch the lower point of the diamond using the small metal lever and the fork-likeknife holder is slid under the diamond (this can be done before scoring or after the knivesbreak). The glass is scored at 2.5, once again not from edge to edge and this time slightlyoff the perpendicular, and broken using the slow and steady movement of the breaking


knob. Once the glass breaks, the clamping head is elevated, the damper is reset (dot tothin line) and the upper holder is released by pulling and rotating the holder knob. Thetwo knives will rest on the fork-like holder which can be removed. They should be left onthe holder and carried to a dissecting microscope. Do not allow them to rest on their sideson a tabletop as the edge may pick up contamination. Caution: Never leave uprightglass knives unattended. Unsuspecting individuals can come along and resttheir hands down on top of the extremely sharp cutting instrument. A cleanplastic (tri-pour) or glass beaker is recommended to cover and protect both yourknives and colleagues.

After preparation of the knives, they must be evaluated for quality. Obviously, poor knivesshould never be used for ultrathin sectioning. Handle the knives carefully so that thecutting edge will not become damaged or contaminated, especially with oil from your skin.Initial evaluation is performed by visual inspection of both knives without a microscope.From the side you will see the shape of a right triangle. The base of the knife will have a1.0mm raised heel. The heel should be an even rectangle with its longest sides parallel.The cutting edge should be relatively straight and level, however, a convex or concave edgeis not uncommon. The convex edge often has the longest usable cutting area. Lookingdirectly at the angled side of the knife, you will notice the sloping conchoidal fractureplane. It is believed that the further to the right this fracture plane exists beforedescending, the longer the usable cutting edge will be. The only way to evaluate a glassknife edge’s quality is to examine it under a dissecting microscope with an overhead lightsource. The setup used for block trimming is ideal. For the best view, a dark background isrecommended. In this case a small square of black photograph mounting board can be cutand placed on the microscope stage. The magnification should be adjusted to allow forviewing of the entire knife edge (~ 30X). The knife should be held with the angled edgefacing outward and the knife set in the shape of a “V”. While looking under thestereomicroscope the glass should be rocked so as to concentrate the overhead light on thevery edge of the knife. The knife edge should lie near the center of the field of view. Whenthe knife edge is focused you should see a broken bright line on the right side of thecutting edge. The broken line is indicative of typical imperfections in the edge which isknown as “frilling”. The left edge of the knife should appear as an unbroken bright linewhich indicates its sharpness and high quality for sectioning. In order for the knife to beusable, at least 1/3 to 1/2 of the left edge should be unfrilled. Knives with quality edgesless than this should be discarded (in an appropriate sharps container to minimize risk tothe maintenance staff). The knife edge can also be evaluated for debris which would leadto the appearance of sectioning artifacts. Do not try to clean the edge of debris using aduster can (compressed air) since particulate and organic matter in the can will damagethe edge. Dirty knives must be discarded with the student exercising greater caution inthe preparation of additional knives. Always prepare more than one good knife in theevent that the knife is damaged or contaminated in transit to the ultramicrotome. Once atthe ultramicrotome, you will not want to backtrack and make more knives.

Once the knives are evaluated, they must be “boated” through the application of a tapetrough/boat. This boat will hold floatation fluid, typically double distilled water, which will


allow the sections to come off the knife edge and float on a liquid surface as opposed tosticking to the dry glass edge. The boat is usually made of mylar adhesive tape and isavailable through some EM supply companies. A suitable substitute would be electricaltape strips which are cut in half (on a clean piece of glass) to reduce the width.

Once a length of tape is cut, the knife glass is placed to it. You will have more control intouching the knife to the tape versus touching the tape to the knife. You should orient theknife to the tape in such a way that only a short length of free tape extends to the left ofthe placed knife. This will minimize the chance of the eventual two free sticky tape endstouching and transferring adhesive to the knife edge. In terms of orientation, the tapemust be exactly even with the upper cutting edge. The lower edge of the tape must beparallel to the lower base of the knife. Once the knife is placed to the tape, the knife can bepicked up and the long free tape end can be wrapped around to fashion a symmetrical boathaving a small gap at the bottom (where the tape meets the angled edge of the knife). Ifthe orientation is not correct, the tape can be carefully pulled off an another attempt canbe made. The more attempts one makes, the greater the risk of contaminating the knifeedge. Excess tape projecting from the back of the knife is cut off using a razor blade andbeing careful not to touch the knife edge. While boating the knife one should be careful notto touch the tape in the region which will actually form the boat. When water isintroduced, oil from the skin will serve to contaminate the floatation fluid and ultimately,your sections. After the tape boat is attached, it must be sealed to prevent leakage usingnailpolish (or molten paraffin wax). The polish should be applied all along the lower tapeedge, especially at the gap on the angled edge of the knife. Excess polish should beremoved from the brush on paper toweling and a thin line run up the two back edges ofthe knife. Do not apply polish to the knife edge. The polish is allowed to dry for about 20minutes at which point, the knife can be used to section on the ultramicrotome.

Since glass is a supercooled liquid, it tends to flow away from the sharp edge and becomedull over time. Ideally the glass knife should be used within 24 hours of manufacture,however, if kept in a storage box and desiccator, it should be useful for a few days. Anotherproblem with the glass knife is that it rapidly dulls with use. Only between 15-30ultrathin sections may be cut before the appearance of visible knife marks which arenoticeable dark, vertical, irregularly spaced lines in the section. These unmarked sectionswill be picked up followed by moving the knife to an unused cutting region. An alternativeto the glass knife is the diamond knife described below.

✥ Diamond Knives

Diamond knives are extremely delicate and expensive instruments which can cost as muchas $5,000.00 based on the length of the edge. Obviously, only experienced microtomistsshould handle a diamond knife since simply touching the edge or cutting a section greaterthan 1.0µm may permanently damage it. The benefits to using a diamond lie in itshardness. Proper care results in a knife that is sharp for years and can cut numeroussections without having to stop and move the knife. The entire edge is sharp which resultsfrom taking a quality gemstone and cleaving it into smaller fragments which are polished


using diamond dust (the actual process is a trade secret of the few companies whichmanufacture diamond knives). The diamond is mounted into a metal holder which is gluedinto a metal trough using epoxy. The need for making glass knives and attaching tapeboats is eliminated.

A common problem in using a diamond is that it is quite hydrophobic which preventswetting of the knife edge. The remedy involves carefully running an eyelash along theinside edge of the diamond with the boat full. If the diamond still won’t wet, a bit of salivaor dilute wetting agent (such as Photoflo 200:1) can be used on the eyelash.

Routine cleaning of the diamond knife involves a gentle stream of distilled water from asquirt bottle to clean the boat and knife edge. On occasion and if sections adhere to theedge, a cleaning stick made of styrofoam or pithwood can be run along the edge, in onedirection only. Never apply forward or backward pressure on the edge. Cleaning sticks canbe dipped in ethanol or even saliva to remove adherent material from the diamond edge.

A recent addition to the types of knives used in ultrathin sectioning is the sapphire knife.As with the diamond knife, the sapphire is permanently mounted in a trough and issuperior to the glass knife. It is however inferior in quality to the diamond knife and isincapable of cutting hard samples such as tooth and bone, without damage to the edge.

✥ Ultramicrotomy

The development of the ultramicrotome has been critical to the formation of high qualityimages of biological material using the TEM. In order for the electron beam to passthrough the specimen, it must be less than 1,000Å thick, ideally between 600-900Å. Toproduce sections thin enough for use in the TEM took years of research into mechanicalengineering of the actual instrument in addition to fixation and embedding materials,blockface requirements and cutting instruments (glass and diamond knives).

When TEM development was taking place in the 1930’s, its use for the examination ofbiological samples was not envisioned. Early investigators who were interested inexamining biological samples with the TEM would attempt to use whole mounts offractured materials with poor result. In 1939, von Ardenne proposed cutting tapering,wedge shape sections whereby a portion of the section would be thin enough for TEMobservation. Modification of a Spencer 820 microtome, used for paraffin embedded LMlevel sections, by Pease and Baker in 1948 resulted in a 1/10 reduction in block advance tothe cutting edge. They also reduced the block face to 1.0mm2 and produced sections 0.3-0.5µm thick. As detailed in the unit on embedding media, Newman et al. (1949) introducedmethacrylates which became the first embedding media of quality for TEM samples. Usinga metal knife which had been sharpened with a new technique, along with an attachedtrough which was used for the first time, Hillier and Gettner (1950) made furthermodifications to the Spencer 820 microtome and produced sections 0.2µm thick. A majoradvance in the field took place in 1950 with the introduction of glass fracture knives byLatta and Hartman. In 1952, Palade used methacrylate embedding, glass knives, a


blockface smaller than 1.0mm2 on an ultramicrotome designed by Claude and Blum andwas able to obtain sections at the useful thickness of 1,000Å. In 1953, the Sorvall “Porter-Blum” MT-1 ultramicrotome was introduced commercially. This instrument was hand-driven and allowed for reproducible ultrathin sectioning by anyone who had the patienceto learn its operation. Needless to say, the first quality TEM photomicrographs ofsectioned biological material appeared at this time.

Since the introduction of the MT-1, numerous improvements in design lead to thedevelopment of the Sorvall MT-2 and 2B with motorized drive, and eventually the MT-9000, currently available from RMC, Inc. LKB (now Leica) was the first company tointroduce an ultramicrotome using the advance principle of thermal expansion. The LKBUltrotome series also had superior lighting and improved specimen blockface to knifeadjustments for greater control in orienting knife to blockface. Current state-of-the-artinstruments include the Leica Ultracut with fiber optic blockface illumination which makeit almost impossible to chop off your blockface, a common problem of the inexperiencedmicrotomist.

Other advances in the field of ultramicrotomy include the work of Peachey (1958) whosuggested that interference colors of sections under a cold (fluorescent) light source canprovide a good estimate of section thickness. He determined that the following sectioncolors relate to their thickness:

60 nm gray60-90 nm silver90-150 nm gold150-190 nm purple190-240 nm blue240-280 nm green280-320 nm yellow

Ultramicrotome Theory

In the design of an ultramicrotome (fig. 3), a rigid cantilever arm is employed to which atrimmed block can be mounted. The trimmed block is held within a chuck. The mostcommon type is a collet which tightens down on the block by turning the threaded collar,usually clockwise, leading to a reduction in the diameter of the circular opening. Otherchucks, such as those used in early LKB ultramicrotomes, resemble nosecones which aresplit in half lengthwise and held together by a hexagonal set screw. Vise-type chucks arealso available with a pair of adjustable flat jaws to accommodate flat embedments. It isimperative that the chuck holds the block securely to avoid vibration during sectioning.

The ultramicrotome cantilever arm must be able to move in three degrees of freedomabout various pivot points. Firstly, the arm must be able to move up and down in an arc;this is known as the cutting stroke since downward movement through a cutting edge


will yield a section. Secondly, the arm must be able to move from side to side in an arc;this is known as the bypass stroke since it allows the blockface to bypass the knife as itis moved to the top of the cutting stroke. Without this motion, the blockface would bedamaged through compression as it hit the back of the knife. In the early Sorvall “Porter-Blum” MT-1, the arm moved within a parallelogram shaped cutout. Modern instrumentssuch as the MT-2 eliminate the side to side motion in favor of a retraction of the arm atthe bottom of the cutting stroke. In the LKB/Leica instruments, the knife stage isretracted electromagnetically. Either way, contact between the blockface and the back ofthe knife is avoided. Finally, the arm must be able to extend linearly along its axis; this isreferred to as the advance since it allows the arm to advance the required minusculeamount to the stationary cutting edge. The amount of advance will determine the ultimatesection thickness. In the development of the ultramicrotome, two methods of advance havebeen used. The first, used in the Sorvall MT-1 and MT-2(B), is mechanical which isdesigned around a vertical pivoting arm which rests on a micrometer lead screw. As thelead screw turns, the lower portion of the vertical arm moves to the back of theultramicrotome, while the upper portion advances out toward the front of the machine.Since the rigid cantilever arm is attached to the upper portion of this pivot arm, it will bemoved forward toward the knife edge. The vertical pivot arm will eventually reach an endpoint and will have to be reset. One should be sure to reset the mechanical advancemachine before mounting a block.

The second type of advance used is thermal which involves the uniform expansion of ametal under uniform heating. This method, used in LKB (now Leica) Ultrotomes forexample, must have provisions for variable heating to control section thickness and acooling fan in order to “reset” the mechanism, since there is a limit of thermal expansionfor any given metal. The arm must be motor driven and electronically controlled. Themechanism must be able to compensate for changes in cutting speed while maintainingsection thickness. By example, if silver sections are cut at 1.0mm/sec and you change thecutting speed to 2.0mm/sec, the machine will have to introduce a point of hesitation intothe cycle to continue to yield silver sections. Without this hesitation, the cycle time wouldbe shortened, therefore, heating time reduced leading to a thinner section.

In terms of cutting speed, the best results are obtained if the cutting speed is uniform(usually somewhere between 1.0-2.0mm/second). Variations in cutting speed was acommon problem in producing good sections using the hand-driven Sorvall MT-1. Manylabs designed a reduction gear to attach to the cycling wheel leading to greater uniformityin cutting speed. Whether mechanical or thermal advance, the introduction of motordrives to the ultramicrotome was a vast improvement.

Another important feature of the ultramicrotome is the stage to which the cuttinginstrument/knife is attached. In some cases the stage is permanently mounted to theultramicrotome (LKB Ultrotome III) while others are removable (Sorvall MT-2). The stagemust be finely machined which will allow for fine control in bringing the knife to theblockface. The stage must incorporate a knife holder (for both glass and diamond knives)and provision for setting the clearance angle, the most important angle of the knife


relative to the blockface, which is initially set at 4˚. There is usually a device/jig to set theproper height of the knife edge in the stage. The stage will also incorporate control for thelateral movement of the knife to select the appropriate cutting area (knife edgeadjustment), rotation of the stage to allow for machine block trimming and mostimportantly, to establish that the lower edge of the blockface be parallel to the knife edgebefore cutting (knife rotation). If not parallel, one side of the blockface will be cut beforethe other. Many thick sections would have to be taken to yield a full blockface which wouldprobably be too large for ultrathin sectioning. The final provision is the stage advance(knife advance) with both coarse and fine adjustments. This control is critical since theknife must be manually moved to the blockface for the first facing cut. Since theultramicrotome arm advances in such small increments, it cannot be expected that thearm would ever reach a stationary knife located some distance from the blockface.Additionally, the cantilever arms are not capable of traveling large enough distances toreach the knife. The knife must be moved to the blockface using the fine controls of thestage. A common problem for the beginner is their lack of patience in advancing the stageto the blockface. The result is a chopped blockface which must be retrimmed.

It should be noted that each control on the stage will have a lock to reduce vibrations.Vibrations can lead to a sectioning artifact known as chatter. Chatter is a regularvariation in thick and thin areas within a section. This produces a uniform dark and lightbanding pattern on the section. Fine order chatter, which cannot be seen under the lightmicroscope, ruins the section by obscuring details. Reduction of vibration is a majorconcern to the microtomist. This can be accomplished by making sure all locks are engaged(chuck, stage, etc.), that the block trimmed is short and broad-based, and that the tablewhich supports the ultramicrotome is sturdy (special anti-vibration tables are availablebut may be unnecessary depending on the room used for sectioning). In general,ultramicrotomes incorporate a massive baseplate in order to lessen the effects of vibration.

Another important feature of the ultramicrotome is lighting. As described earlier, afluorescent light source is used to produce section interference colors for thicknessdetermination. Good lighting is also important for the critical advance and initialalignment of knife edge to blockface. It is recommended that the position of the light beadjusted regularly during advance of the knife to the blockface for the best possible view.Modern instruments incorporate fiber optic lighting on the blockface which leads to a moreaccurate approach and fewer chopped blockfaces.


Fig. 3

Microtome ArmBlock

Collet Chuck

GlassKnife

Clearance Angle(2-5°)

Water Level

Water Surface

Ultra-thinSections in

Ribbon

Knife Edge

Mic

roto

me

Arm

Blo

ckCo

llet

Ch

uck


MT-2B Ultramicrotome Sectioning Procedure

The following describes the procedure to follow when sectioning with the Sorvall “Porter-Blum” MT-2B ultramicrotome. Many of the general steps can be applied to the proper useof any microtome on the market with a noted improvement in lighting over the MT series.Failure to adhere to the procedure as outlined may result in frustration and many choppedblockfaces.

INITIAL SETTINGS & PROCEDURES

• Press Reset Button• Upper Thickness Pivot Knob at 10 (10 x 10Å = 100Å)• Thickness Wheel (front left side) at 12-14 (1200-1400Å)• Cutting Speed at 1.0mm/sec• STAGE: Fully Retracted with ALL LOCKS OFF - Do NOT Place on MT-2B at this time!

THICK SECTIONING PROCEDURE

• Mount trimmed block and orient with fluorescent light fully extended out!Firstly, obtain a fully reflective blockface using loosened “arc” adjustment andhand tighten thumbscrew (B&L binocs set at highest magnification).Secondly, orient bottom of blockface parallel to “ground” using chuck rotation andhand tighten (This adjustment is not critical since knife edge parallel can beobtained using stage knife rotation).Use tools to completely tighten chuck (NEVER OVERTIGHTEN ANYTHING).

• Mount Knife in Holder (Be sure front of knife is flush with aluminum guide plate).• Mount Knife Holder in Stage and Lock at 4˚.• Push Light Source back in and Mount Stage to MT-2B (use stage rotation lock on left).• Stage should be pushed forward completely until it stops.• Check Position and Duration and set if necessary (observe from side of setup).• Using lowest magnification setting on B&L binocs, locate block pyramid and knife edge.• Focus on knife edge and bring pyramid into focus by rotating cycling wheel clockwise.

THIS MUST BE DONE OFTEN OR YOU WILL CHOP YOUR BLOCKFACE !!!• Coarse advance the stage (knife) to the blockface. Increase the binocs mag as blockface

and knife edge get closer. Always refocus on the knife edge, THEN, bring theblockface into focus by cycling (focus the “cutting” stroke and not the “retraction”stroke).

• Your goal is to get the knife edge and blockface in focus together in the same field ofview at the highest binocs mag (“3” = 45X).

• Position your knife to face the block near the center of the knife edge and gently LOCK.• Advance closer using the coarse control until you feel you might chop your blockface.• After cycling the blockface to the lowest point in the cycle, fill your boat with clean

distilled water using a drawn micropipette or syringe, to a slight positive meniscus.Level and clean the water surface using Ross Lens Tissue (the water should be of auniform reflective gray without any dark shadows near the knife edge).


• Lock out coarse advance and go to fine advance using thumbscrew (NEVER TIGHTENSCREW WITH STAGE IN FULLY RETRACTED POSITION). Only the largediameter advance barrel should turn with each line equal to 1.0µm increments.

• Critically focus on the knife edge and cycle the blockface back into sharp focus.• Fine advance without cycling until extremely close to knife edge. Determine if bottom

of blockface is parallel to knife edge. If not, carefully rotate stage until correctedand gently LOCK stage rotation.

• When critically close, begin to interleaf cycling with fine advance with a final reductionin advance to 1.0µm per cycle prior to your first section coming off.

• You may note a light reflection of the knife on the blockface (see diagram). Thereflection (best seen in a faced block) indicates you are at least 10.0µm from theface.The reflection will shrink and finally disappear when you are 10.0µm from the face:

• Your goal is to take 4-6 full face thick sections (1.0µm green) by manual cycling.• With the blockface at the lowest point in the cycle, pick up some thicks using your

eyelash and place them in a drop of water at the center of a clean microscope slide.• Allow to air dry and observe using the phase contrast microscope for the presence and

quality of tissue.• Proceed to thin section if all checks out.

ULTRA-THIN SECTIONING PROCEDURE

• With block in lowest point of cycle, refill and clean and level boat (Ross Tissue).• Retract with fine advance (50µm at this step and 10-20µm thereafter).• Cycle blockface into critical focus with knife edge (should see reflection).• Unlock and move knife edge just inside of quality area of knife as was determined by

prior evaluation. Gently RELOCK.• Fine advance until reflection disappears then with cycling and 1.0µm advance until the

first section is cut.• Lock the stage advance and START the MOTOR (depress red button).• Note section colors (probably purple) and reduce thickness using side (NOT PIVOT)

thickness wheel until PALE GOLD/SILVER is obtained.WALK AWAY - LET MACHINE DO ITS JOB !!!

• After 15-20 sections are cut or until noticeable vertical knife marks appear, STOPMOTOR.

• Isolate floating sections using the eyelash and pick up on the dull side of 200-400 meshGRIDS (use 200 mesh for ribbons, 300-400 mesh for loose sections (undesirable).Hold slightly bent grid in jeweler’s forceps and touch dull side down on top offloating sections. Turn grid dull side up and blot dry on filter paper. Unlock forcepsand place grid on grid gripper or on edge in grid box.


• Follow ultra-thin sectioning instructions above and obtain more silver sections.REMEMBER TO UNLOCK THE STAGE ADVANCE LOCK BEFORE RETRACTINGTHE STAGE AND MOVING THE KNIFE FURTHER LEFT !!!

BE PATIENT & GOOD LUCK !!

Final Ultramicrotomy Comments

Some additional comments are warranted with regard to the MT-2B procedure which wasjust outlined. Under “initial settings and procedures”, the required steps should beobvious. Recommended initial cutting speeds are usually in the 1.0 to 2.0mm/sec range.The stage should be carefully checked to ensure that it is fully retracted otherwise, youcould easily ram the knife into the blockface when the stage is first mounted to theultramicrotome.

Under “thick sectioning procedure”, the initial blockface orientation is critical. The goal isto obtain an initial full-face thick section manually. This is impossible unless theblockface is properly oriented. In order to accomplish correct orientation, the properlighting on the face is important. With the MT series, the fluorescent light must be fullyextended so that the blockface is illuminated from the front. After blockface orientation,the light must be placed in a rear position in preparation for advancing the knife to theface. When mounting the knife in its holder, be careful not to break the tape/nail polishseal. Make sure that the knife seats on top of the height adjusting set screw and notbehind it. If the knife is not flush with the front aluminum guide plate, the clearance angleyou set will be inaccurate. A feature of the MT-2B is position and duration controls (theMT-2 did not have these controls). They provide fine control over the ultramicrotome’scutting range and are best demonstrated at a slow cutting speed (0.33mm/sec). At thisslow speed you will notice the actual cutting range of the instrument. The actual rangeexists over the position and duration of this slow rate of cantilever arm travel. Above andbelow the cutting range, the arm moves faster. Obviously, you want the cutting range tocoincide with the knife edge. Looking from the side of the stage/block-arm setup, one mustensure that the slower range of blockface/pyramid travel is coincidental with the knifeedge. If not, the position of the cutting range can be raised or lowered and/or the duration(length of cutting range) can be lengthened or shortened.

Initial facing of the block should be done at or slightly to the right of the center of the knifeedge. The left side should be reserved for ultrathin sectioning. When filling the boat, it isimportant that the blockface be positioned at the lowest point of the cycle. The indicator ofa proper water level is a uniform reflective silver-gray surface appearance. If the block isat a high point in the cycle, it will cast a shadow on the water surface and you will beunable to determine the proper level. Relative to trough fluids, double distilled water in asterile centrifuge tube works best. Some investigators use a low concentration of 1-3%acetone or ethanol to facilitate wetting of the knife edge. Although rarely seen with glassknives, failure of distilled water to wet the diamond knife edge is common. Carefully


running an eyelash along the inside edge of the diamond is often a solution, especially ifthe eyelash has been dipped in a wetting agent (Kodak photoflo) or even saliva.

Only experience and practice will enable you to determine when to begin fine stageadvance and ultimate interleafing of advance with cycling in order to avoid ramming yourblockface. The reflection of the knife edge in the blockface is the best indicator of distance,however, it is often difficult to see in a hand trimmed blockface. Once thick sections areobtained, picking them up becomes difficult for many beginners. An eyelash which isbrought up quickly from underneath a group of sections works well. If the eyelash provestoo difficult, a syringe tip or shaved toothpick can be tried. The toothpick should be dippedin acetone or you may introduce a large amount of debris into the boat. The thick sectionswill be used to verify the presence of tissue in the section along with its quality andorientation.

Under “ultrathin sectioning procedure”, you must be sure to first unlock the stage advanceand then retract the stage before moving the knife to section in a new area. The knife/stage will be fine advanced with a good reflection of the knife edge in the faced block. Thereflection will also aid in the determination of parallel relative to the knife edge andbottom edge of blockface. Once the first semi-thick section is cut, the stage advance lockmust be engaged to prevent chatter. Once the motor is started, ultrathin sectioning willbegin. Reduce the side thickness control until silver sections are obtained. If the block wasproperly trimmed, a ribboning of sections will occur. Once knife marks appear, the motoris stopped with the blockface at the lowest point in the cycle and the sections are pickedup on the dull side of a grid. Pickup is easily accomplished by simply touching the dullside of the grid to the floating sections. To protect the tape boat seal, a bend is put in theedge of the grid (do not attempt to bend formvar coated grids). With the edge of thegrid locked into the jeweler’s forceps, it is laid flat, dull side down, on filter paper. Theforceps are then raised to create an approximate 45˚ angle with the filter paper surface.The bent grid held by the forceps is put aside and the eyelash is used to separate longribbons into ribbons of 15 to 20 sections and isolate them in the center of the boat (isolatedsilver sections can also be rounded up at the center of the boat if ribbons are not formed).The grid is picked up and using the unaided eye, positioned over the boat in a flat,horizontal orientation. Looking through the binocs, the grid can be arranged so that thesections/ribbon will be centrally located after pickup. The grid is gently lowered to thesections so as not to disrupt the surface tension forces and taken away. Do not submergethe grids! The dull side of the grid is used for a number of reasons. Since the dull side isrough in comparison to the polished (shiny) side and the sections themselves are rough,they adhere better to the dull side of the grid. Another advantage is that you establishuniformity and always know what side your sections are on and on the dull side, you canactually observe the reflective sections after you collect them (fig. 4).

After sections are collected, grids are blotted dry on edge (see post-staining procedure-Chapter 3) and stored dull side up on a “grid gripper” (also known as a grid mat) to airdry, after which they can be post-stained. The grid gripper prevents grids from attachingto the lid of the petri dish via static electricity. The safest place to store grids is on edge in


a grid box. Serial sections collected on slot or hole grids should never be put on a gridgripper or blotted flat down on filter paper.

Fig. 4


Troubleshooting Guide to Ultramicrotomy

When an instrument capable of cutting sections on the order of 600-900Å is used, itbecomes obvious that a number of problems may be encountered. The following sectionprovides information on troubleshooting the most common problems associated withultrathin sectioning. Although at times this process can be quite infuriating andfrustrating, the key to mastery of the ultramicrotome is practice. The more problemsencountered at the outset, the better equipped one becomes at finding solutions. Studentsmay initially get lucky and achieve good results on their first attempt but it is unlikelythat they will be able to produce consistent quality results unless they encounter some ofthe problems which are described below.

• Inability to cut any sections:a. microtome advance not resetb. specimen/blockface too largec. poor embedding or soft blockd. poor knife (not evaluated correctly) or improper clearance angle settinge. water level in trough too lowf. vibrations in roomg. blockface gets wet due to high water levelh. faulty ultramicrotome (check that all locks are tight)

• Variations in section thickness from one section to anothera. blunt/dull knife edgeb. incorrect clearance angle (usually needs to be increased)c. incorrect cutting speedd. soft blocke. drafts or temperature changes in the roomf. faulty ultramicrotome (check that all locks are tight)

• Knife marksa. imperfections or contamination of the knife edge - knife poorly evaluated

• Chattera. resin and tissue hardness are not properly matchedb. poorly trimmed block - excessively high/tall, not well supportedc. locks not engaged - looseness leads to vibrationd. cutting speed may be too highe. clearance angle may be too highf. blockface too large (approaching 1.0 sq. mm)


• Compression (can use organic vapors of chloroform or heat to flatten sections)a. cutting speed too highb. incorrect clearance anglec. poor knife edged. soft block

• Folded/Wrinkled sectionsa. poor knife edgeb. cutting speed too highc. water level too lowd. soft blocke. incorrect clearance angle

• Sections pulled down back of knifea. water level too highb. cutting speed too slowc. clearance angle too small

• Ribbons not straight or not formeda. poorly trimmed blockb. two sides of blockface parallel to knife edge are not parallel to each otherc. water level in trough not correctd. incorrect cutting speede. blunt/dull knife

• Holes in sectionsa. poor infiltration of resin into tissueb. air bubbles in resin/tissue - vacuum infiltration is recommended to avoid this

• Cannot see sections as they come off the knife edge/cannot achieve proper trough levela. adjust/move illuminationb. blockface not at lowest point in cyclec. leaky boat seal

It becomes obvious from a glance at these troubleshooting tips that many problems can besolved by manipulating cutting speed and/or knife clearance angle. LKB/Leica hassummarized a methodical approach to varying these parameters. They suggest starting at4˚ and a speed of 2mm/sec. If the results are unsatisfactory, they recommend changing thespeed to 1mm/sec, then 5mm/sec. If good results are still not obtained, a change inclearance angle to 1˚ is recommended (don’t forget to retract your knife sufficiently)starting once again at 2mm/sec and moving to 1mm/sec, then 5mm/sec. A final step wouldinvolve changing the clearance angle to 7˚ using the identical cutting speeds as previouslynoted.


At all steps of ultrathin sectioning, contamination is to be avoided. The most commonproblems arise when oil is not removed from razor blades used in trimming and glassstrips are not adequately cleaned with a non-filming soap and rinsed well enough.Additionally, as glass knives are prepared they become contaminated from oily fingers andtrough tape adhesive. Trough fluids must be pure double distilled water and one must becareful not to contaminate the fluid by introducing dirty instruments such as grids,forceps and eyelashes into the boat. If the trough fluid is dirty, section contamination isinevitable. Once sections are collected on grids, they must be stored in a dry, dust freeenvironment such as a grid box. Care should be taken not to drop the grids. Keep a layerof Ross lens tissue under the work area as you handle the grids. If you should happen todrop the grid, at least it will be onto a non-linting paper. Be especially careful when post-staining which is a common source of section contamination.

Materials Required for Ultrathin Sectioning

The following is a complete list of all the materials required in the EM lab to conductultrathin sectioning and all related procedures such as block trimming and glassknifemaking.

• Block Trimminga. Properly embedded tissue within resin blockb. Small (50ml) beaker containing acetone for the removal of razor blade oilc. Double edged razor blades (cleaned with acetone prior to use)d. Ross lens tissue (used to dry and clean blades before and during use)e. Microtome chuck (collet, etc.) to hold blockf. Trimming base plate for supporting the chuck while trimmingg. Pliers (to squeeze block out of BEEM capsule)h. Small metric ruler to estimate size of trimmed blockfacei. Dissecting microscope with overhead illuminator (such as Bausch & Lomb)

• Glass Knifemakinga. Plate glass strips (LKB/Leica recommended)b. Liquid soap (non-filming such as Liquinox)c. Distilled water for final rinsingd. LKB/Leica Knifemaker (7800 series or more recent model)e. Dissecting microscope with overhead illuminator for knife edge evaluationf. Mylar boat tape (or strips of electrical tape which are cut in half lengthwise after

attachment to a clean strip of plate glass)g. Nail polish for sealing the boatsh. Razor blade for cutting excess boat tape


• Ultramicrotomya. Trimmed block (0.25 - 0.5mm on the longest side with two sides parallel)b. Glass fracture knives with attached and sealed boats (2-4 knive are recommended

with at least 1/3 to 1/2 quality cutting edge on left side as previouslyevaluated)

c. Ultramicrotome (complete with microscope head, stage, glass knife holder, coldlight source, and any required accessories)

d. Double distilled water in sterile centrifuge tube (place in test tube rack)e. Clean micropipette or tuberculin syringe to fill boatf. Eyelash to pick up thick sections and to orient thin sections in boatg. Ross lens tissue (to clean and regulate meniscus level in boat)h. Acetone cleaned 200, 300, 400 mesh grids (on clean filter paper in a covered petri

dish)i. Jeweler’s forceps with an o-ring lockj. Filter paper in covered petri dish to blot grid dry after section collectionk. Grid gripper in covered petri dish for temporary storage of grids (quality grids

should later be moved to a grid box for permanent safe storage)


Chapter 3 - Post-Staining

In order to form an image, enhancement of contrast (differences in optical or graindensity) is necessary, especially when an ultrathin section is considered. In lightmicroscopy, colored dyes/stains are utilized in order to improve contrast and impart colordifferences. In the preparation of biological samples for the TEM, electron dense, heavymetal stains are used.

The most common staining employed is double post-staining, meaning that two stains areused after the sections are collected on grids. Another technique which is readily found inthe literature involves en bloc staining of tissue blocks before the blocks are embedded inthe epoxy resin(s). Typically, tissue blocks are stained en bloc using aqueous 0.25% to 4%uranyl acetate solutions for 5-60 minutes just prior to dehydration in ethanol (afterosmium tetroxide post-fixation, tissues should receive a buffer wash).

Post-staining involves the use of the salts of the heavy metals, lead and uranium.Caution should be used in handling these heavy metal stains since they areextremely toxic, especially in powdered form. Uranyl salts are low-levelradiation emitters. Disposal of heavy metals should be done in accordance withlocal regulations.

The usual stains employed are uranyl acetate, followed by lead citrate. Uranyl acetate andlead citrate react will many cellular components including proteins and nucleic acids. Ifone does not stain with uranyl acetate, the nuclei are most affected with a noticeable lackof contrast due to weak or no staining of the nucleic acids contained within. Both stainsare said to be positive stains since they increase the density of the structures underconsideration as opposed to the background. In negative staining, the backgrounddensity is increased relative to the structures (as in the negative staining of virus particlesusing 1-2% phosphotungstic acid - PTA). The preparation of stain solutions is describedbelow.

✥ Uranyl acetateA 0.5% aqueous solution of uranyl acetate is prepared by dissolving 0.2g of uranyl acetatein 40ml of distilled water in an Erlenmeyer flask (50ml) and gently mixing over a 10minute period. The solution will be clear and yellow with no notable sediment. Thesolution is photosensitive and should be stored in the dark at room temperature. Thesolution may be filtered or centrifuged just prior to use, however, this is usually notnecessary so long as you take the solution from the center of the given volume. It shouldbe noted that uranyl acetate solutions can also be prepared in methanol (saturatedsolution), ethanol or acetone and in volumes less than 40ml. Many investigators willprepare small volumes as needed to reduce the possibility of contamination.


✥ Lead CitrateThere are a number of formulations for lead citrate with the most widely used developedby Reynolds (1963) and Venable and Coggeshall (1965). In the Reynolds formulation, 1.33gof lead nitrate and 1.76g of sodium citrate are added to 30ml of CO2-free, double distilledwater in a 50ml Erlenmeyer flask and mixed regularly over a 30 minute period. At thispoint, 8ml of 1N (4%) NaOH is added with an obvious “clearing” of the solution uponmixing. Finally 12ml of the CO2-free, double distilled water are added with gentle mixing.The final working solution should be placed into sterile centrifuge tubes. The tubes shouldbe filled to capacity to minimize CO2 bearing air from coming in contact with the solution.The tubes containing the solution should be stored in the cold at 4˚C and centrifuged for aleast 5 minutes just before use. The final pH of the solution should be checked and ideallybe alkaline at 12 or higher.

The main problem associated with the use of lead citrate is contamination. Lead citratereadily reacts with atmospheric CO2 to form a crystalline precipitate known as leadcarbonate. On your sections, lead carbonate deposits are extremely electron dense leadingto large, unsightly dark staining artifacts which obscure detail. One should avoidbreathing directly on the lead citrate solutions. As you stain, you should breathe out of theside of your mouth and also minimize the time of lead exposure to the air.

The Venable-Coggeshall formulation allows you to prepare smaller quantities of thesolution as it is needed. In this recipe, 0.01 to 0.04g of lead citrate are added to 10ml ofCO2-free, double distilled water in a centrifuge tube along with 0.1ml of 10N (40%) NaOHand mixed.

In order to prepare CO2-free distilled water, it can be autoclaved and sealed right afterremoval or it can be boiled for at least 10 minutes and then sealed while hot. Allow thewater to come to room temperature before solution preparation.

✥ Post-staining Procedure

Grids can be post-stained within 15 minutes following section collection, after the gridshave air-dried. The following procedure should be used with care taken not to contaminatework areas with toxic heavy metal stains (fig. 5).

• Using a squirt bottle, sprinkle water on the table surface.• Take a precut 4" by 4" square of Parafilm and pull along the water surface to produce asmooth, flat sheet without air bubbles. The sheet should be spread smooth with theprotective cover (imprinted with the product name) in place as to prevent water fromcontacting the parafilm surface. Once flattened, the protective cover is removed and thefilm is covered with a Petri dish lid.• Place 10-15 pellets of NaOH (caution - caustic, causes severe burns) at the top of theparafilm sheet and recover with the petri dish lid. The NaOH should be wetted slightlyand will serve to absorb atmospheric carbon dioxide, reducing the possibility of leadcarbonate formation and contamination.


• Using a Pasteur pipette, place as many drops of aqueous Uranyl acetate stain in ahorizontal row to correspond with the number of grids to be stained, not to exceed five (5)drops per setup. If one holds the tip of the pipette near the surface of the parafilm and hasgood control over the pipette bulb, the diameter of the drops can be adjusted. The dropsshould be just slightly larger in diameter that the 3.0mm grids themselves. The UA dropsshould be placed in a row just below the NaOH pellets without coming in contact with theNaOH.• Using a different pipette, place an identical row of CO2-free distilled water drops beloweach drop of the UA without placing them too closely together. Then, skip a row (leavingroom for a row of lead citrate) and form two (2) more identical rows of the CO2-freedistilled water.• Without direct breathing on the solution or staining setup, lay out the row of lead citratedirectly below the first distilled water row using a clean, new pipette. Recover with thepetri dish lid.• STAINING: Place a grid section (dull) side down in a drop of UA using jeweler’s forcepsand stain for 15 minutes with the petri dish cover in place. From 1 to 5 grids can bestained simultaneously. After 15 minutes the first grid in UA can be carefully picked up onedge and blotted on edge with a filter paper circle. The filter paper circle should be placedbetween the tines of the forceps which hold the grid. The circle can now be more accuratelycontrolled in its movement to contact the edge of the grid. A small amount of stain will beabsorbed onto the filter paper. The blotted grid can be placed into the water drop belowthe UA drop for rinsing (30-60 seconds). Repeat for each grid as necessary (up to a total offive).

As blotting commences, the filter paper circle should be rotated to a clean area andeventually discarded. The tines of the forceps can be cleaned regularly throughout theprocess by blotting them on a clean filter paper region. After the first water rinse, bloteach grid on edge as described and move them into the lead citrate stain for up to 5minutes (DON’T BREATHE!!). After the lead citrate, the grids can be blotted and movedthrough two water rinses for approximately 1 minute each. After the final water rinse, thegrid is blotted on edge and placed section (dull) side up on a grid gripper or stored on edgein a grid box to dry. After post-staining, grids can be observed using the TEM within 15minutes, when they have dried.

Be extremely careful not to damage the grids during staining. Since the grids are handledquite often during staining, it is common for beginners to mangle their grids. This is notdesirable considering all of the work that has come before the staining process.

If you encounter a large amount of staining artifact, you can try using a two setup methodwhere one setup contains UA followed by three rows of water (without NaOH pellets) andthe other contains NaOH pellets, a row of lead citrate and three rows of water. This setupminimizes lead citrate exposure to the air. Some investigators will use a rinse row of0.01N NaOH below the lead citrate in this setup, however, some loss of lead staining mayresult.


Fig. 5

Uranyl Acetate (15 min)

Distilled Water (30-60 sec)

Lead Citrate (2-5 min)



Parafilm (4"x4")

NaOH Pellets

Petri Dish


Chapter 4 - Grids & Grid Supports

✥ Grids

Grids are typically 3.0mm in diameter and can be made of any non-magnetic metal suchas gold, platinum and nickel. The most common metal used in the manufacture of grids iscopper. They come in a variety of mesh sizes (referring to the number of bars per inch) andmesh patterns. Common grid mesh sizes are 200, 300, 400, up to 1000. It is recommendedthat large sections in ribbons be collected on 200 mesh or larger size grids. The ribboninghelps support the sections and larger mesh sizes translate into a larger viewing area (lesssection regions covered by grid bars). Isolated sections should be picked up on 300-400mesh grids in order that they be supported and not wrap around grid bars. Square meshgrids are routine, however, hex-mesh and other shapes are available. Finder-grids are alsoavailable with reference coordinates to help you relocate previously examined sections.Open hole and slot grids, which must be coated (see grid supports below), are useful forserial sectioning.

Grids should be cleaned before use by sonicating them for 5 minutes in a small beaker ofacetone. After sonication, the excess acetone is poured off and the beaker is placed upsidedown on a 9cm diameter filter paper circle in the large diameter lid of a petri dish. As theacetone evaporates, the clean grids will drop to the filter paper surface. Use the other halfof the petri dish to cover the grids and label the their type and size with a marker.

✥ Grid Supports

The use of modern epoxy resins in the embedding of biological samples for the TEM hasreduced the need for grid support films. The three-dimensional polymerization of epoxyresins with the addition of the proper curing agents and heat (60˚C) or UV, results in anembedment which is resistant to damage from solvents and heat, including the electronbeam. Ultrathin sections are therefore very stable under the electron beam. Embeddingmedia which were used in the past for TEM, such as the methacrylates, were unstableunder the primary beam with the resulting ultrathin sections requiring a support. Evenwith the use of epoxy resins, grid supports may be necessary. A good example involves theprocess of serial sectioning. Serial sections allow us to follow a particular structurethrough many ultrathin sections in order to develop an understanding of its three-dimensional architecture. Since grid bars could block the ability to view the structure(s) ofinterest, typical square-mesh or hexagonal-mesh grids are not employed for the collectionof serial sections. Instead, an open slot or circle grid is used (see below). The large openarea cannot support sections, therefore, a grid support must be prepared.


Square Mesh Grid Hole/Circle Grid Slot Grid

The most common type of support used today is either plastic - Formvar (polyvinyl formal)or carbon films or a combination of both.

Formvar Films

Working solutions of formvar in ethylene dichloride (0.25%) can be purchased directlyfrom EM supply companies or you can prepare the solution yourself by placing 0.25g offormvar powder into 100ml of ethylene dichloride and gently mixing. The solution shouldbe clear with no sediment and stored in a brown bottle out of direct sunlight and heat.CAUTION: Ethylene dichloride is flammable and is a potential carcinogen. Whenready for use the solution should be transferred to a clean coplin jar.

Supplies:0.25% Formvar solution in Coplin jarclean 400ml plastic beaker (tri-pour)Glass Microscope SlidesRoss Lens TissueSingle-edged Razor BladeLiquinox soap (non-filming)Distilled waterGrids (to be coated)Jeweler’s Forceps

The glass slides are cleaned using Liquinox soap, rinsed in distilled water and allowed tostand on end and air dry. The plastic beaker is then filled completely (about overflowing)with distilled water and returned to the work area. A clean glass slide is dipped slowlyinto the formvar solution and withdrawn. The end of the slide is placed on Ross lens tissueto remove the excess solution and allowed to air dry for a few minutes (some individualsgently wave the slide to hasten drying). The slide is now scored with a single-edged razorblade just inside the periphery of the glass slide and across the slide into three to foursquare regions (fig. 6). The slide is picked up (scored side up) and slowly introduced intothe distilled water of the plastic beaker at a very shallow angle (20-30˚). Prior to theintroduction of the formvar coated slide, the distilled water surface is cleaned by pulling asheet of Ross lens tissue across the water surface, as was done to clean the water surfaceof the glass knife boat used in ultramicrotomy. Just before placing the formvar coatedslide into the distilled water, one can breathe on the scored surface. “Frosting” the surface

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in this way is believed to facilitate stripping of the formvar off of the glass slide. Underfluorescent lighting, you should observe floating formvar “sections” in the beaker. Thesefloating films should appear of a uniform silver color under fluorescent lighting. If thefilms are dark gold, purple, etc., the solution must be cut with the addition of ethylenedichloride. If the films are too thin, you can add more powdered formvar to the solution.Formvar films which display a variety of colors (rainbow) may be contaminated and a newsolution should be freshly prepared.

The greatest difficulty in working with these plastic films involves their stripping from theglass slide when placed into the distilled water. Exceptionally clean slides are not ideal forthis purpose. You can also experiment with slides of different manufacturers as someprove better than others. To facilitate formvar stripping, a thin and even coating of oilfrom the skin (usually best when taken from alongside the nose) can be applied by indexfinger to one surface of the slide (make sure you know which surface is oil-coated since thisis the side you will want to score). One can also use a thin coating of saliva on one side ofthe slide. Both techniques work well to strip the formvar from the slide and it should benoted that contamination of the formvar does not occur as a result.

Once the formvar films are cast on the water surface, the slide can be dropped to thebottom of the beaker. Using the fine forceps, grids can be carefully placed, dull side down,on the floating formvar film. Concentrate the grids at one end of the film withoutoverlapping them. The final technique involves collecting the coated grids, dull side up(with the formvar surface on top). Some individuals use a large container such as afishtank to submerge the films from above with a glass slide. The slide is swept throughthe water in a large “U” fashion and is removed with the coated grids on top of the slide.Others use a rapid “slap and flip” technique with a glass slide to collect the coated grids.The simplest method involves placing a clean glass slide or wax coated cardboard stripexactly perpendicular (90˚) to the floating formvar film, in the region without any placedgrids and gently submerging the slide using a straight downward force (fig. 7). This filmwith the coated grids just rolls up on the slide with the grids in the correct orientation(dull side up). The slide is removed from the water and allowed to air dry. Once dry, thegrids are picked up from the slide surface and either used immediately or stored for lateruse.

Fig. 6 - Formvar Coating on Glass Slide

Glass SlideFormvar Coating

Razor Blade Score Lines


Fig. 7 - Collection of Formvar Coated Grids

Razor Blade Score Lines

Clean DH2O Surface

Slot Grids (Dull Side Down)

Glass Slide


Carbon Coating

Carbon films are strong and extremely stable under the electron beam. Additionally, theycan be made very thin and are essentially electron transparent. Carbon coating can becarried out alone or on top of plastic films in order to provide increased stability. Someinvestigators also “sandwich” various support films around the ultrathin sections (plastic-sections-plastic, plastic-sections-carbon).

Carbon coating is usually conducted in a high vacuum evaporator. The high vacuumcreated in the bell jar is required so that air molecules will not interfere with theevaporated carbon and lead to an uneven coating. Two pure carbon rods are prepared sothat one is sharpened to a point and the other is flat on end. The rods are locked into thetwo suitable electrodes in the evaporator with the pointed end of one rod in direct contactwith the flat end of the other. Beneath the carbon rods are the grids you wish to coat on aglass slide and a white porcelain plate with a drop of low vacuum oil or immersion oil atits center. The bell jar is evacuated to 10-5 Torr and voltage is applied to the electrodes. Atthe pointed carbon rod tip, it becomes white hot and carbon is evaporated. The carbon fallsto evenly coat the grids. Thickness can be determined by the darkness of the porcelainplate in comparison to the central oil spot. Due to the presence of the oil, the area beneathit will not darken but rather, remain a bright white. The desired color of the indicatorplate is described as a light tan/brown which results in a 400-700Å thick coat(fig. 8).

Since carbon does not adhere well to naked copper grids, an initial coating of plastic suchas formvar is recommended. If desired, the plastic coat can later be dissolved away inchloroform and ethylene dichloride.

Carbon coating attachments are also available for low vacuum sputter coaters (seediscussion of sputter coating in Unit 2 on SEM Sample Preparation). Carbon coating ofSEM samples over metallic coating (gold, etc.) is required when elemental analysis isbeing conducted.


Fig. 8 - Carbon Evaporation Setup

oil

grids

electrode

carbon rods

Bell Jar


Unit 2 - Preparation of Biological Samples for SEM

Since the same high vacuum environment (10-5 Torr) is required in order to generate anelectron beam in the conventional SEM as is required for the TEM, the observation ofliving samples is not usually possible (environmental ESEM’s and differentially pumped,low vacuum SEM’s may permit the observation of living samples). However, because theSEM is utilized primarily in the observation of surface features, the tedium of ultra-thinsectioning, necessary to TEM specimen preparation, is avoided. Depending on the natureof the surface of interest, a sample might be prepared simply by adhering it to analuminum support or stub and examining it in the SEM. Of course, such a sample wouldhave to be hard and conductive, characteristics rarely found together in biologicalspecimens. Common examples of hard surfaces include chitinous insect and crustaceanexoskeletons, lignified cellulose cell walls of plant material/wood, calcium carbonate shellsof molluscs/bivalves such as oysters and clams, silicon frustules (shells) of diatoms, andcomplex calcium and phosphorus hydroxyapatites which comprise part of the non-livingmatrix of bone. A problem exists in that these and other biological samples are usuallyinsulators which are incapable of emitting an adequate signal when contacted with theSEM primary electron beam. Lack of adequate signal leads to an inferior quality image(low SNR). In addition, there are many occasions when one wishes to examine a soft tissuesample which would readily degrade under the high vacuum operating environment of theSEM, not to mention, be subject to the forces of autolysis and decomposition. Anotherfactor to consider is whether the visualization internal structure using a SEM is desirableor even possible.

Since ultra-thin sectioning is not required for SEM samples, they can typically be larger(recall that TEM tissue blocks had to be minced to a thickness no greater than 0.5mm inone dimension to ensure complete infiltration of fixatives and embedding media). Whendealing with soft tissues, the tissue pieces should remain somewhat small (~2-3mm3) toallow for complete penetration of the primary fixative and prevention of internal collapseand resultant artifacts in surface morphology (shape). Also of importance is the handlingof small samples and microbes. The following chapters shall be divided into thepreparation of a variety of samples including hard tissues/structures, soft tissues, internalstructures, microbial samples and SEM samples prepared for the TEM for SEM/TEMcorrelation.

UNIT 2 – PREPARATION OF BIOLOGICAL SAMPLES FOR SEM 59

Chapter 5 - Hard Tissue Preparation

As stated earlier, hard tissue samples can include chitinous insect and crustaceanexoskeletons, lignified cellulose cell walls of plant material/wood, calcium carbonate shellsof molluscs/bivalves such as oysters and clams, silicon frustules (shells) of diatoms, thenon-living matrix of bone and teeth. Such samples may have adherent soft tissue and/orsurface debris associated with them. This material should be removed using as delicate atechnique as possible. Surface lint and dust may be easily removed with compressed air(care should be taken since some compressed air sources contain particulates which couldinduce surface damage). In addition, surface debris can be rinsed away using distilledwater or an isotonic physiologic buffer solution (the need for an isotonic solution isquestionable since hard tissues are unlikely to shrink or swell osmotically). In the case ofsoft tissue adhering to samples such as bone, the soft tissue can be dissolved away byboiling in 28% ammonium hydroxide followed by subsequent rinses in distilled water.

CAUTION: Ammonium hydroxide must be opened and used in a fumehood. Due to its extremely high vapor pressure, a bottle of 28%ammonium hydroxide opened in even a well ventilated roomwill quickly saturate the atmosphere with toxic ammonia.Inhalation could prove to be fatal.

Following cleaning, which may not be necessary, hard tissue samples are mounted to astandard 15mm diameter, aluminum specimen mount or stub, using an appropriateadhesive (types of suitable adhesives will be covered later in this unit).

Depending on specimen conductivity, or the usual lack of it when dealing with biologicalsamples, and the desired SEM imaging voltage (low ≤ 5kv or high > 5kv to 25kv), themounted samples will then be given an ultra-thin (100 - 200Å) conductive coating of ametal such a gold, platinum, palladium, or gold-palladium. Since most samples aregenerally insulators and high voltage is usually selected for maximum signal generationand the resultant high quality images, the norm involves the application of this conductivecoating. The techniques and procedures of sputter coating and vacuum evaporation will becovered in detail later in this unit, in conjunction with soft tissue preparation.

It should be noted that even though hard samples are inherently more durable than theirsoft tissue counterparts, care should be exercised in handling these samples so as not toinduce artifacts. This is especially true of samples such as small insects which should begently attached to the specimen stubs using fine forceps. The use of liquid adhesives forsmall samples should be avoided since the liquid has a tendency to creep up the sides ofthe specimen and envelope it. This is not a problem if one wishes to study glue!


Chapter 6 - Soft Tissue Preparation

In order to examine soft tissue samples in “as near to life-like condition as possible”,chemical fixation is required. Without chemical fixation, post-mortem autolysis anddecomposition would distort even the surface of the specimen of interest. Even with thesurface “stabilized” through conductive coating, internal degradation would lead to glaringartifacts of surface morphology. In addition, chemical fixation will permit observation ofinternal structures using cryo (cold) techniques and allow for the correlative preparationof SEM samples for the TEM via epoxy resin embedment and ultra-thin sectioning.

The preparation of soft tissue samples for examination in the SEM begins in an identicalmanner to the preparation of samples for the TEM (see Unit 1 - Preparation of BiologicalSamples for TEM). A generalized protocol follows:

•Primary Aldehyde Fixation•Buffer Wash•Secondary Osmium Tetroxide Postfixation•Optional Buffer/DH2O Wash•Dehydration Series (Ethanol/Acetone)•Intermediate Fluid Series (Freon TF/113)•Critical Point Drying (in Transitional Fluid - liquid CO2 or Freon 13)•Mounting (adhesive on 15mm dia. aluminum stub)•Conductive Coating (Sputter Coater or Vacuum Evaporator)

Firstly, the tissues of interest must be excised and placed into the primary fixative. As forTEM preparation, we typically use a combination method of in situ and immersionfixation. The tissue blocks for SEM preparation can be somewhat larger since thealdehydes (primary fixative) can penetrate through at least 3mm of sample. Tissue blocksare therefore best minced to the dimensions of 2-3mm3. If strips of tissue are cut, theyshould be no thicker than approximately 2-3mm on one of the sides.

Minced tissue blocks can be transferred to vials containing the primary aldehydefixative and remain for one hour. Once again the best single aldehyde choice is the doublyreactive, cross-linking, glutaraldehyde. As for TEM sample primary fixation, we use a 3%concentration of glutaraldehyde carried in the proper isotonic buffer vehicle (for most softmammalian tissues, 0.2M phosphate buffer, pH 7.2-7.4 is ideal). The chemical action ofaldehydes (stabilization of the cellular protein matrix) and other fixatives along with thepurpose of buffer solutions and their preparation are covered in the chapters on TEMspecimen preparation.

Once the primary fixation is complete, three buffer washes of ten minutes duration eachare conducted to remove unbound aldehyde and prevent the undesirable precipitatereaction with the secondary fixative, namely, osmium tetroxide.


Following the buffer washes, the samples are placed into the secondary fixative, osmiumtetroxide. The OsO4 will react mainly with unsaturated lipids and impart an increase inconductivity to the biological sample. The OsO4 is also carried in the same buffer vehicleas for the glutaraldehyde. You will recall that the benefit to osmium tetroxide fixation forTEM was the introduction of an electron dense stain (for phospholipid membranes andother osmophilic structures) for added contrast. In SEM, the generation of a signal fromthe surface is of paramount concern. Incorporation of the heavy metal osmium to thesample allows for increased surface signal emission when contacted by the primaryelectron beam of the SEM. Later in this unit, I will discuss how osmium incorporation canbe enhanced by the addition of agents such as tannic acid and thiocarbohydrazide(TCH), so much so, that conductive surface coating may be avoided.

An optional buffer or distilled water wash of two changes for ten minutes durationeach may be performed, although, I have not observed any artifacts arise due to theomission of this step.

Next, the tissue must undergo complete dehydration, but not for the same reason asTEM sample dehydration. Residual water in the tissue would cause surface tensioncollapse as it dried. For this reason, the common process of critical point drying - CPD(or some alternative) is employed in the preparation of samples for the SEM. The CPDprocess, which is carried out under liquid carbon dioxide, would be ineffective if waterremained in the tissue block. A complete description of CPD theory and procedure is foundlater in this unit. By contrast, you will recall that samples are dehydrated for TEM inorder that the epoxy resins infiltrate the tissue blocks (epoxies are not miscible withwater). Both acetone and ethanol (EtOH) are common dehydrating agents used. Sinceshrinkage of tissue blocks and the resultant surface distortion is undesirable for SEMspecimens, the dehydration schedule is more gradual for SEM, starting at 30% EtOH. Theusual ascending series is 30%, 50%, 70%, 95% EtOH for ten minutes each, followed by100% EtOH for two changes of ten minutes each with the vials being filled to capacity asusual. The preparation of such a dehydration series using 95% (not 100%) ethanol hasbeen described in the unit on TEM specimen preparation.

The final steps in the soft tissue protocol will involve the preparation of tissue blocks forcritical point drying which has traditionally been performed in either liquid carbondioxide or liquid freon (Freon 13). Usually, before the critical point drying process, tissueblocks are passed through an ascending intermediate fluid series of Freon TF/113 sinceit is miscible with both ethanol and the transitional fluid used in critical point drying. Thefreon is diluted with 100% ethanol, not water, in order to produce the concentrationseries. The usual ascending series is 30%, 50%, 70%, 95% Freon TF/113 for ten minuteseach, followed by 100% Freon TF/113 for two changes of ten minutes each.

As the scientific community has become more aware of the environmental impact of freonson the degradation of the protective ozone layer (which partially shields the earth fromharmful UV radiation), alternatives to its use have been discovered and will be consideredfollowing the discussion of critical point drying. A common past alternative to Freon TF


was amyl acetate with its characteristic strong, banana-like odor. It was typically usedas an intermediate fluid between ethanol and liquid CO2. Due to its toxicity, it shouldalways be purged out of a critical point dryer into a fume hood. A benefit to its use wasthat one always knew it had been completely purged from the critical point dryer be themarked absence of its trademark odor.

The process of chemical fixation of biological soft tissues have been carried out in a liquidenvironment. If in the final step, these tissues are simply allowed to air dry, tremendoussurface tension distortion will occur leading to obvious surface artifacts. It should be notedthat water can exert a surface tension force of over 2,000 psi. In order to prevent thissurface tension damage, the technique of critical point drying (CPD) has been utilizedsince 1968.

The principle of CPD involves an understanding of phase (solid-liquid-gas) boundaries,especially, for our purposes, the boundary between liquid and vapor. Every fluid possesseswhat is known as a critical density (Dc) at which the boundary or interface (the actualliquid surface) between the liquid phase and the vapor phase becomes indistinguishable.At first, when a fluid is introduced to a sealed container, an equilibrium exists betweenthe liquid and vapor phases. In order to attain critical density, this equilibrium must beshifted to the vapor phase through heating and the related increase in pressure. Criticaldensity (Dc) is attained at the critical temperature (Tc) and critical pressure (Pc) of thegiven fluid. Therefore, to eliminate the interfacial boundary between liquid and vapor andavoid the associated surface tension force, the fluid must be elevated to its criticaltemperature and pressure.

Since the critical temperature and pressure of water is excessive, at 374˚C and 3,184 psirespectively, and would damage delicate biological soft tissues, other liquids typicallyserve as transitional fluids (so named because they make the transition between liquidand vapor phases). The most common of these is liquid carbon dioxide (LCO2) and liquidfreon 13. Due to its higher price and negative impact on the environment, liquid freon hasbecome the less popular of the two transitional fluids. Liquid CO2 has a criticaltemperature and pressure of 31˚C and 1,073 psi, respectively, compared with liquid freon13 at 28.9˚C and 561 psi. The liquid CO2 is introduced to the critical point dryer at thehigh pressure (600 -800 psi) of its storage tank. The storage tank is specially ordered witha siphon tube which takes the liquid CO2 from the bottom of the tank since as the tank isemptied, CO2 gas rises and collects at the top of the tank.

CAUTION: Liquid carbon dioxide is under high pressure and is very cold.Use care in handling the tanks and opening the tank valve. Theuse of a regulator is not required, however, the tank pressureis between 600-800 psi. Make sure the hose between the CPDand the tank is threaded and tightened properly, and is a hoserated for high pressure applications (a minimum burstpressure rating should be printed on the hose - the hose usedin this lab is rated at 17,000 psi). In addition, the storage tank


should be chained or strapped to a wall or a tabletop toprevent it from accidentally falling and rupturing. Under itshigh pressure, the tank could act as a missile causing seriousinjury. Finally, CO2 is a colorless, odorless gas and should bevented from the CPD into a fume hood to prevent asphyxiationas it displaces the normal atmosphere.

✥ Critical Point Dryer Operation

The CPD is a thick walled metal chamber which can withstand pressure in excess of 2,000psi. The chamber is constructed of a metal of excellent heat conductive properties such asbrass, copper or bronze. The chamber is usually machined to accommodate some type(s) ofspecimen holder(s) which will retain the tissue blocks throughout the procedure. Thechamber also includes three high pressure needle valves, the inlet valve (for theintroduction of the LCO2), the drain valve (to drain off the intermediate fluid - freon TF/113) and the vent valve (to vent the CO2 gas). Additionally, the CPD chamber must havesome provision for heating the transitional fluid. In the more expensive models, there is anelectrical heater. In the least expensive models, the entire CPD chamber is simply loweredinto a container or bucket of hot water. The main drawback to this method is that residualexternal water on the unit might contact the tissues as they are removed, rehydratingthem. Our Pelco Jumbo Dryer (same as the Polaron Jumbo) is designed with a waterjacket just external to the drying chamber. Hot and/or cold water can be routed throughthe jacket by means of plastic tubing attached to a faucet. A temperature gauge is addedin order to monitor the water temperature, although, the only required gauge is a chamberpressure gauge. The Pelco CPD is equipped with a safety valve which is designed torupture at 2,000 psi.

CAUTION: The CPD is a high pressure device which if used improperlycan lead to serious injury or death. Follow all manufacturerdirections carefully. If equipped with a thick quartz viewwindow (as in the case of the Pelco Jumbo Model), it MUST BEEXAMINED FOR CRACKS BEFORE EACH USE!!! NEVERREMOVE the protective Lexan shield, if so equipped, justoutside the view glass - it is there for your safety!! NEVERLOOK DIRECTLY INTO THE VIEW GLASS (use a mirror if youare curious) - if it were to break, the high pressure wouldproduce many high velocity, very sharp projectiles! Ideally,the CPD should be located in a fume hood. For obvious reasonsstated above, the CPD is alternatively known as “the BOMB”.

In the routine operation of the Pelco CPD, the unit is placed into the fume hood and thehigh pressure hose is connected tightly to the liquid CO2 tank. The inlet water hose isattached to the faucet and the outlet water hose is run into the hood sink. The drain hose(optional) is also run into the hood sink. The heavy rear door is removed using the steelrod tool. If this is the first CPD run of the day and the bomb is at room temperature, you


will not have to cool the unit by running cold water through the water jacket. If the bombhas been heated, it will have to be cooled to approximately 20˚C before use. It is often a goodidea to perform a “dry run” of the unit to check it for any leaks (valves, window and reardoor dowdy seals may require tightening) - it is better to test the unit than to sacrifice goodtissue blocks which will ultimately dry down with surface tension collapse in a bomb whichcannot maintain pressure! All three needle valves should be closed clockwise. The valve onthe liquid CO2 tank can now be opened.

Tissue blocks in their second change of 100% freon TF/113 can now be loaded into the CPDboat. Most CPD units come with a device to contain any number of specimen holders. ThePelco unit comes with a three reservoir/channel boat into which some nine specimenbaskets, which resemble sewing thimbles, can be loaded. The boat has a large rearstainless steel pin which interfaces with a hole in the rear door of the CPD. At the front ofthe boat is a spring loaded valve which when inserted into the CPD, opens to allow theintermediate fluid to drain out. Firstly, the CPD boat is filled with freon TF/113. At thispoint, the tissues are transferred from their vials to the tissue baskets. Sine most tissueswill float in 100% freon TF/113, they are easily transferred by pouring the vials contentsinto the baskets over a sink.

CAUTION & REMINDER: All potentially toxic chemicals used in thefixation process should be handled using disposable gloves andwearing goggles, ideally within a fume hood!

Once transferred to the baskets, the baskets are quickly placed into one of the three CPDboat channels which are full of the intermediate fluid, freon TF/113, to prevent prematuredrying down through a liquid interface. Sheets are available (fig. 9) to identify the specifictissue which occupies a specific basket in the boat. Once a channel contains three baskets,a wire mesh cover is placed on top of the baskets to prevent tissue loss in the bomb. TheCPD boat can hold nine baskets at a maximum.

Once the boat is loaded with tissue baskets, the boat is carefully carried over to the bomband gently inserted. A slot on the rear underside of the boat must align with acorresponding slug in the rear floor of the CPD chamber. The central hole of the rear dooris lined up with the stainless steel pin of the boat and the rear door is threaded clockwiseuntil tight. Use the steel rod tool to tighten the rear door and prevent leaks. Your tissuesare now ready to undergo critical point drying.

With the tissues properly loaded into the CPD and with all valves closed, the threevalves will be used (or as I like to call it, juggled) in order to introduce the liquid CO2 andflush out the freon TF/113 (fig. 10). Initially, the upper inlet and vent valves are opened.Do not be alarmed at the amount of noise coming from these high pressure needle valves.The vent valve must be opened to allow for the escape of gas and permit the requiredvolume of transitional fluid to enter the bomb. You should be able to observe the liquidCO2 enter the bomb and rise to a desired level above the boat. As the liquid CO2 isentering the bomb, the lower drain valve must be opened to flush out the freon TF/113


under pressure.

Fig. 9

At first, a clear fluid will be observed flowing down and out of the drain line, followed by asolid CO2 “snow”. Allow the solid CO2 to exit the drain line for a few seconds, ensuringcomplete flushing out of the freon TF/113, and then close the drain valve. Using the inletand vent valves, allow the liquid CO2 to reach a volume just above the top of the boat, thenclose both valves. At this point, you should not hear any “hissing” which would be typicalof a seal or valve leak. The pressure gauge should be holding steady at 600-800 psi (Theactual liquid CO2 tank outlet pressure).


Fig. 10

Now, hot water can be flowed into the water jacket. The flow rate should be moderate (asviewed directly at the outlet hose) and the water temperature should be approximately 40-45˚C. As time passes and the heat is conducted from the water jacket to the CPD chamber,the temperature gauge and pressure gauge will rise accordingly. At approximately, 29˚Cand 1,000psi, you will observe a turbulence at the liquid CO2 interface, followed by the

PEDESTAL

SUPPORTCOLUMN

SAFETYVALVE

DRAIN VALVE

WATEROUTLET

VIEWGLASS

REARDOOR

VENTVALVE

INLETVALVE

WATERINLET


instantaneous disappearance of the liquid interface at critical temperature and pressure(once again, the use of a mirror is recommended for viewing this process vs. lookingdirectly into the CPD view glass). When this phenomenon occurs, at critical temperatureand critical pressure for the given fluid, the density of the liquid phase equals the density ofthe vapor phase (known as the critical density) and the boundary between the two phasescan no longer be distinguished. The tissues within the bomb have been critical point driedwithout surface tension distortion.

Even though the critical temperature and critical pressure for liquid CO2 is 31˚C and 1,073psi, respectively, the bomb chamber is heated to a somewhat higher level (approximately40˚C and 1,500 psi) to prevent recondensation of the CO2 vapor. The bomb is held at thislevel for a few minutes then slowly (approximately 100 psi/min.) vented to theatmosphere using the vent valve. Rapid venting would result in recondensation of the CO2vapor and wetting of the dried tissue blocks. At atmospheric pressure, the CPD boat iscarefully removed to a dry location by slowly unscrewing the rear door of the CPD. Thetissue baskets are uncovered and removed to a clean, dry work area. Small (7ml) specimenvials are ideal for supporting the tissue baskets at the work area as long as they are dryinternally. The tissue blocks, which are obviously dry and somewhat brittle in appearance,are now ready for adhesive mounting to specimen stubs.

✥ Alternatives to Critical Point Drying

Due to current concerns with the effects of chlorofluorocarbons (CFC’s) such as freon onthe environment (protective atmospheric ozone), the tendency has been away from the useof intermediate fluids and transitional fluids and critical point drying.

Fluorocarbon Drying

In an alternative to critical point drying, a solid fluorocarbon such as Peldri II, has yieldedgood results. In the use of Peldri, this solid fluorocarbon is heated to its melting point andheld until use in its liquid state. The tissue blocks are brought through the dehydrationseries and placed into the liquid Peldri. The mixture is taken off the heat source andallowed to solidify. Once solid, the samples are placed into a low vacuum environment andthe solid Peldri sublimates to the vapor state without passage through a liquid interface.Dry tissue blocks are mounted and coated as usual.

Organo-Silicon Compounds

A more recent alternative to the use of freons as intermediate fluids and transitionalfluids for CPD is the use of the organo-silicon compounds, Tetramethyl silane (TMS)and Hexamethyldisilazane (HMDS). Samples are simply placed into these compoundsafter routine dehydration. Although liquids at room temperature, these agents evaporatefrom the sample surface without the usual surface tension forces exerted by other liquids.Air drying is therefore performed using these compounds without the surface artifactsproduced when using other liquids, such as water. Air drying using these compounds has


been reported at both room temperature and 60˚C (in an incubator).

Adhesive mounting of dry tissue blocks to the typical, 15mm diameter, aluminumspecimen stub is a rather simple matter. Firstly, the specimen stubs should be previouslycleaned by sonicating them in a beaker of acetone for 5-10 minutes. The stubs are removedand allowed to air dry on a sheet of lint-free cloth. A variety of adhesives have been usedfor mounting SEM specimens to the stubs. They include liquids such as cyanoacrylates(‘crazy glue’), rubber cement, low resistance contact cement, silver paint (for a conductivecontact to the stub), and Pliobond (manufactured by Goodyear - it is easily solubilized inacetone and ideal for removal when taking SEM tissue blocks to resin embedment andsectioning for TEM). The main drawback to liquid adhesives is their tendency to creep upthe sides of a small sample and quite possibly cover the surface in the glue. To preventthis, a long time favorite for the attachment of small samples has been double sided scotchtape. Even better, we have found that adhesive transfer tabs, manufactured by the Averylabel company, to be ideal for the adhesive mounting of most every SEM sample. Theseglue tabs are simply lifted off their backing sheet and placed in the center of the stub.Downward pressure is applied as indicated on the surface of the tab, and the tab isremoved, leaving a thin layer of adhesive behind.

Tissue blocks, or any other sample, are carefully handled with jeweler’s forceps and placedinto the adhesive of choice. A stereo-microscope can be used if specific tissue orientation isimportant to your study. By example, if you wish to examine small intestinal villi, youshould image the sample under the stereoscope to ensure that the villi are facing upwardversus placing them into the glue.

The final step in the preparation of soft biological samples for SEM, not to mention hardsamples, is the application of a thin (approximately 100-200Å thick) conductive coat tothe surface of interest. This is done since most biological samples are insulators and wouldyield a poor surface signal (secondary electron emission) when contacted with the primaryelectron beam. In addition, these samples would readily degrade under the high voltage(>5kv) beam. Aside from high voltage beam damage, conductive coating is required toprevent what is known as charging artifact; a buildup of negative surface charge whichserves to repel the primary electron beam and also prevents the loss of secondaries fromthe surface. Uncoated samples could be imaged under low voltage (≤5.0kv) conditions,however, with a resultant loss of image quality (low SNR).

✥ Conductive Coating

Currently, there are two techniques utilized in the modern EM lab for putting a thinconductive coat on a sample. They are Vacuum Evaporation and Sputter Coating.Each method will be described below.


Vacuum Evaporation

In this technique, a high vacuum evaporator is required. This device (see fig. 8) wasoriginally described in the chapter on TEM Grids & Grid Supports (Chapter 4) under theheading of carbon coating. The main difference here is that in place of mounting the flatand pointed carbon rods into the evaporator electrodes, a pointed (bent into a V-shape)piece of tungsten wire is mounted. At the bend/point of the tungsten filament, a short, thinpiece of gold, platinum or gold/palladium is wrapped. Directly under the tungsten filamentis the rotating mechanical specimen stage with the provision for insertion of specimenstubs. Once the filament and stubs are set up, the bell jar is put in place and the volume isevacuated to high vacuum (10-5 Torr). At this point, the stage motor is energized and therheostat is advanced until the tungsten burns white hot and the gold, gold/palladium, etc.melts (a noticeable bead of molten metal forms) and then evaporates to thinly and evenlycoat the sample under the rotating stage and high vacuum conditions. The bell jar isbrought back to atmospheric pressure and the samples removed from the stage. Careshould be exercised when removing the samples. Be sure not to touch the embrittledtungsten wire which might break and cause large particulate contaminants to rain downupon your unremoved samples. After use, the evaporator bell jar environment must berestored to high vacuum – all high vacuum systems should always be maintained at highvacuum.

CAUTION: The process of evaporating these metals should be observedthrough a piece of thick, dark glass. The intense brightnesscould permanently damage the retinas of your eyes.

Sputter Coater

The sputter coater operates at low vacuum (10-2 Torr) using a small rotary pump toevacuate a Pyrex cylinder. The system is typically maintained at atmospheric pressure.Operation of a modern sputter coater, such as the Denton Desk II in this lab, is quitesimple and somewhat automated. The one requirement for the sputter coater is a tank ofultrapure (at least 99.9% pure) argon gas. Such tanks of inert gas are under high pressure(approximately 2,500 psi when full) and must be regulated down (this tank must also bechained to a table or wall near the coater unit). A single or double stage regulator isrequired to bring the outlet pressure to 5 psi. A plastic or tygon tube connects the tank/regulator to the sputter coater. Before use of the coater, the argon tank valve must beopened and the regulator pressure knob advanced to 5 psi. If there is a valve after theregulator as is our case, it must be fully opened (all valves typically open clockwise unlessotherwise noted). You should understand the argon is not yet flowing into the sputtercoater chamber. It is flowing down the plastic tubing and is stopped at an automaticsolenoid valve. The sputter coater has a hinged head (fig. 11) into which runs a highvoltage (1-3kv) cable. This cable interfaces with a thin gold (or platinum, palladium, etc.)foil target which serves as the source of the conductive heavy metal. A replacement goldtarget currently costs approximately $400.00.


Once the stubs with mounted samples are placed on the platform/stage under thesputterhead, the cylinder is evacuated to low vacuum conditions (50 millitorr) while theultrapure argon gas is introduced. A negatively charged high voltage field (1-3kv) isapplied to the gold source cathode/target.

This negative field ionizes the argon atoms to positive ions as per the equation below:

Ar˚ ➞ Ar+ + e-

Due to electrostatic attraction, the positive argon ions are accelerated to the negative goldsource cathode. The argon ions randomly strike the gold target forcibly causing theejection of gold atoms from the target at various angles. These gold atoms build up aneven conductive coat on the surface of the samples. A permanent magnet attracts strayelectrons which result from the ionization of the argon atoms, and sends them to ground.These energetic electrons could interact destructively with the surface of the sample if notremoved. It should be noted that sputter coating can be conducted under atmosphericconditions, however, ionization of many normal atmospheric components, even water,results in the formation of a number of highly reactive ions/radicals which would readilydamage the surface of our delicate biological samples. Argon is chosen since it is an inert,stable or noble gas. Once samples have been coated, the chamber is returned toatmospheric pressure and the stubs are carefully removed.

NOTE: After samples have been conductive coated, they should be storedin a desiccator toprevent absorption of atmospheric water vapor. Ifdesiccator chambers are not available, small desiccators can be fashionedout of Petri dishes. The lower rim should receive a thin layer of vacuumgrease or petroleum jelly. Small holes can be punched or burned into largeBEEM capsules which are then filled with silica gel and capped. Thecapsules (1-2 per dish) are placed into the Petri dish. They need to bechecked at least weekly as the silica gel hydrates (you will observe a colorchange from deep blue to pink to clear as the silica gel hydrates).Hydrated silica gel can be collected and baked in an oven at 350˚F to driveout the water for reuse.


Fig. 11

Denton Desk II Operation

This modern sputter coater can coat samples reliably in under five minutes (comparedwith at least 30 minutes for the vacuum evaporator). It has LED readouts for pressure (inmillitorr), current (in milliamps) and time (countup or countdown). It can be run inmanual or auto timed modes as desired. Generally, a test run is desirable to establishnormal operating parameters such as pressure and current before introducing yourvaluable sample stubs. The routine operation follows:

• Prepare argon tank and regulator (5 psi outlet pressure) as described previously.• Switch on main circuit breaker on rear of machine.• Open sputterhead and place stubs on top of stage.• Close sputterhead making sure it seats properly on the seal - vacuum grease is usually

not necessary.

To Rotary Pump

STAGE

SPECIMEN

PERMANENT MAGNETGOLD TARGET CATHODE

HIGH VOLTAGE CABLE

SPUTTERHEAD

Argon Inlet


• Switch on MAINS (red switch) on the right side of the machine - the rotary pump willcome on.

• Observes pressure gauge and allow to drop below 100 millitorr (when mains are firstenergized, the OFF button flashes, then glows steady - it MUST glow steady toproceed to the next step).You may need to adjust the pressure using the pressure control knob near thepressure LED.

• Depress the SPUTTER button - you will hear a “pop” which is the opening of thesolenoid valve to admit the argon gas. At this point, the SPUTTER knob lightshould be glowing steady. This step may fail at first attempt. In that case, allow thepressure to once again drop below 100 millitorr and try again - you may need toadjust the pressure knob.

• Adjust the pressure using the pressure control knob to 50 millitorr.• For manual timing, depress the manual start button & for automatic timing depress the

timed start button - in either case high voltage is applied to the gold target and aplasma field is observable as a bluish ring just below the target. The manual timercounts up and the auto timer counts down from whatever you preset the time for(up to a maximum of 999 sec).

• Using the current control knob, quickly regulate the current to 45 milliamps - if theplasma field weakens and begins arcing/flashing, the pressure needs to be increasedusing the pressure control knob.

• Once the desired time has elapsed, press stop for manual timing (auto timing will autoshut off the high voltage after the preset time has elapsed).

• Turn off the MAINS switch - the solenoid valve will shut and the vent valve willautomatically open to restore atmospheric pressure.

• Open sputterhead and remove stubs to a desiccator for storage.• Repeat as needed for coating of each stub (the stage accommodates about six 15 mm

diameter stubs).

As noted earlier, a manual test run with no stubs present is desirable to preset thevacuum and current at optimum levels. Maintenance of the sputter coater is rather simpleas well. Each year, the rotary pump oil and mist filter should be changed. When the goldfoil target breaks through continued usage, it is easily replaced by removing the fourthumbscrews which hold the meshwork cage in place. Four screws are removed from theretaining ring which holds the circular foil in place. The old foil is removed and the newfoil put in place using the retaining ring and meshwork cage. On occasion, the pyrexcylinder should be cleaned of sputtered gold using 95% ethanol. If heavily coated, a goodmetal polish such as Wenol or Pol, followed by 95% EtOH works well.


✥ Comparison of TEM and SEM Soft Tissue Protocols

When one compares the preparation of soft tissue for the SEM and the TEM, a number ofobvious differences becomes apparent. The following table summarizes those similaritiesand differences which were detailed in the chapters on SEM and TEM specimenpreparation:

TEM Protocol

3% Glutaraldehyde (Phosphate buffer) – 1hr.

Buffer Wash – 3 X 10 min.

1% OsO4 (Phosphate buffer) – 1hr.

Optional Buffer/DH2O Wash – 2 X 10 min.

70%, 95%, 100% EtOH – 2 X 10 min. each

Propylene Oxide (P.O.) – 3 X10 min. each

1 Part P.O. : 1 Part Epoxy Resin – 1hr.

Pure Resin – Vacuum Infiltrate – 1hr.

Embed in BEEM Capsules

Cure @ 60˚C – 48hrs.

Ultra-thin Section

Mount Sections on Grids

Heavy Metal Staining

SEM Protocol

same

same

same

same

30%, 50%, 70%, 95% EtOH – 1 X 10 min. each100% EtOH – 2 X 10 min.30%, 50%, 70%, 95% Freon TF – 1 X 10 min. ea100% Freon TF/113 – 2 X 10 min.Critical Point Dry in Liquid CO2 (31˚C, 1,073 psi)

Sectioning Not Required

Mount Tissue Blocks on Stubs

Conductive Coating


Fixation ScheduleSEM Mammalian Soft Tissue Protocol - In-situ and Immersion fixation - 2-3mm3 tissue blocks


Schedule Duration

3% Glutaraldehyde (0.2 M phosphate buffer, pH 7.4) 1 hour

Buffer Wash 3 x 10 minutes each

1% OsO4 (buffered as above) 1 hour

Buffer or DH2O Wash (optional) 2 x 10 minutes each

30% Ethanol 1 x 10 minutes each 50% Ethanol 1 x 10 minutes each 70% Ethanol 1 x 10 minutes each 95% Ethanol 1 x 10 minutes each 100% Ethanol (fill vials completely) 2 x 10 minutes each

Optional LN2 Cryofracture - between 1st & 2nd 100% EtOH - see description in text!

30% Freon TF/113 1 x 10 minutes each 50% Freon TF/113 1 x 10 minutes each 70% Freon TF/113 1 x 10 minutes each 95% Freon TF/113 1 x 10 minutes each 100% Freon TF/113 2 x 10 minutes each

Critical Point Dry - in LCO2

Adhesive Mount on Aluminum Stubs

Sputter Coat (Gold) - 50mT, 45mA 30-45 seconds


Fixation Schedule WorksheetSEM Mammalian Soft Tissue Protocol - In-situ and Immersion fixation - 2-3mm3 tissue blocks



3% Glutaraldehyde (0.2 M phosphate buffer, pH 7.4)

Buffer Wash

1% OsO4 (buffered as above)

Buffer or DH2O Wash (optional)

30% Ethanol50% Ethanol70% Ethanol95% Ethanol100% Ethanol (fill vials completely)100% Ethanol (fill vials completely)Optional LN2 Cryofracture - between 1st & 2nd 100%EtOH - see description in text!

30% Freon TF/11350% Freon TF/11370% Freon TF/11395% Freon TF/113100% Freon TF/113100% Freon TF/113Critical Point Dry - in LCO2


Sputter Coat (Gold) - 50mT, 45mA


Chapter 7 - Alternative SEM Specimen Preparation

A number of alternatives and enhancements to the “typical” biological soft tissue protocoldescribed above, are available. The following are the most common alternate procedures.

✥ Uncoated Specimens

Conductive samples require no coating, which even at 100Å thickness can obscure surfacestructures. Conductive samples should typically be imaged at high voltages (20-25kv) totake advantage of the increased signal to noise ratio (SNR) and yield a higher qualityimage.

In order to view the non-conductive biological sample in an uncoated state, a low voltagebeam (≤5.0kv) is required so as to minimize beam damage to the sample. Unfortunately,the use of a low voltage beam will drastically reduce the generation of the secondaryelectron signal from the surface and produce a poor image (low SNR). It is interesting tonote however that even though the uncoated, low voltage image is of inferior quality incomparison to an identical coated sample imaged at high voltage, the amount informationwhich is hidden through conductive surface coating is significant.

In terms of uncoated biological samples, investigators have prepared them in a variety ofways for introduction to the SEM. In the simplest example, fresh specimens have beenutilized. Such samples would have to be quite resilient in order to hold up under the highvacuum environment. Organisms with external shells and exoskeletons, such as insects,are good examples. In addition, cellulose containing botanical samples, including pollengrains, might be observed “fresh”. One should avoid samples which contain abundantwater since dehydration surface tension artifacts will occur in addition to unacceptablylong pump down times for the SEM.

An alternative to ambient temperature fresh specimens is the use of a cryogen in order tofreeze the sample prior to insertion in the SEM. Common cryogens employed includeliquid freon 13 and liquid nitrogen. The sample, which is cleaned in water or buffer asnecessary, is mounted onto a stub and plunged into liquid nitrogen. In order to maintainits frozen state, the SEM must be fitted with a cold stage which is held at liquid nitrogentemperature. It should be mentioned that this technique differs from freeze drying whichis conducted under vacuum conditions with samples that are typically primary fixed inbuffered aldehyde and cryo-protected in glycerol or dimethyl sulfoxide (DMSO) beforebeing plunged into a liquid/solid cryogen “slush” and “dried” in a freeze drying unit undervacuum. The cryoprotectant will minimize damage induced through the formation of icecrystals in the extreme cold.

In another alternative to uncoated specimen preparation, there exist a number of osmiumenhancement agents which serve to concentrate additional osmium tetroxide into thesoft tissue sample rendering it conductive, without the need for surface conductive coating.


Examples of such osmium enhancement agents include Tannic Acid andThiocarbohydrazide (TCH). Thiocarbohydrazide has been used successfully in this andmany other labs in a protocol known as OTO and the alternative, OTOTO, with “O”indicating the osmium tetroxide solution and “T” indicating the TCH solution. In the OTOprocedure, the typical primary aldehyde fix, buffer washes and osmium tetroxide (“O”)postfix is conducted. This is followed by a distilled water wash, then, 1% aqueous TCH(“T”) followed by 1% aqueous osmium tetroxide (“O”). This is followed with a distilledwater wash and continuation of the usual protocol dehydration series. The OTOTOprocedure adds an additional 1% aqueous TCH and 1% aqueous osmium tetroxide step tothe OTO procedure. Critical point drying is conducted for soft tissues followed by adhesivemounting, but without the need for conductive coating. Samples can be imaged under highvoltage conditions with good stability and signal generation.

✥ Cryofracture Technique

Since the SEM is limited to the imaging of surface features, techniques have beendeveloped that allow the viewing of internal structures. One of the most useful of these forthe internal imaging of biological/cellular structures is cryofracture. In this technique, atissue block is immersed in liquid nitrogen (LN2) for a few seconds and then fracturedon a chilled brass or copper plate/disk using a pre-chilled, single-edged razor blade.

A basic procedure which has yielded excellent results in this lab involves performing thecryofracture step between the two 100% ethanol changes of the dehydration series. Thevial, maximally full of 100% EtOH and containing the tissue blocks are brought to thecryofracture station along with forceps, another vial filled with 100% EtOH (and labelledwith the tissue type and a reference to cryofractured samples - for example, kidney (c),with the (c) designating samples which have been cryofractured), and a new acetonecleaned, single-edged razor blade. The cryofracture station (fig. 12) consists of the lowerportion of a Petri dish, into which is placed a copper or brass (excellent heat conductors)disk and the clean razor blade. When ready, the Petri dish will be filled with LN2 to apoint even with the top surface of the copper disk. If your lab has the large LN2 tanks withcryo hoses, the Petri dishes can be filled directly from the hose or by transferring a smallamount of LN2 to a small dewar vessel for pouring into the dish. In this lab, we maintain a10 liter dewar of LN2 which has an integral dipper for transferring small volumes of LN2to the Petri dish.

CAUTION: Liquid Nitrogen is extremely cold at -150˚C. It can causeinstantaneous frostbite injury. Goggles must be worn toprevent eye contact and cryogloves are recommended. Itshould be noted that prolonged contact with the cryogen dueto the Leidenfrost effect is more damaging to your tissues thana momentary surface contact with exposed skin. You wouldhave a much greater chance of frostbite damage if the LN2were to be trapped against the skin in a pair of gloves.NEVER touch anything which has been cooled with LN2!!


The next steps should be performed quickly yet carefully as the liquid nitrogen evaporatesrapidly. A tissue block is selected from the 100% EtOH vial using the jeweler’s forceps andsubmerged into the LN2 in the Petri dish. The block is held for a few seconds and thenplaced on the surface of the now chilled copper disk. The cold razor blade is used tofracture the tissue block using a deliberate vertical, downward force. The fractured piecesare then transferred to the empty 100% EtOH, cryo labelled vial using the cold forceps.These tissue blocks can be processed the rest of the way as usual.

In alternative protocols to the above, a cryoprotectant such as glycerol or DMSO can beutilized. In a protocol devised by Tanaka (1981), DMSO was used as described below:Following the osmium tetroxide postfixation, samples are transferred to 25% DMSO(DMSO is diluted with buffer), then 50% DMSO for 30 minutes each. The samples arethen cryofractured as described above, except that they are put into 50% DMSO after thecryofracture has been completed (not 100% EtOH). A buffer wash is performed followed bythe usual dehydration series. Using this protocol, Tanaka was able to view well preservedinternal cellular ultrastructure such as mitochondrial cristae and Golgi membranes.

Fig. 12

PETRI DISH

LIQUID NITROGEN

COPPERDISK

FORCEPS & TISSUE BLOCK


✥ Microbial Specimen Preparation

Microbes such as bacteria, protozoa and others present a unique challenge to preparationfor observation under the SEM. The main problem involves our inability to observe theseorganisms with the unaided eye. How can we be sure that samples are present in thespecimen vials? How can we avoid sucking them up with our pipettes while decanting offsolutions? The most important developments in the field of microbial specimenpreparation for SEM have involved the design and utilization of a number of containmentstrategies for the organisms of interest. A variety of commercial and homemade sampleholders have been used over the years. Keeping containment in mind, it is obviouslycritical to have some idea as to the dimensions of the organism you wish to fix.

In the past, both nucleopore and millipore filters, along with polycarbonate membranes,have been used for such specimens as bacterial cells (~0.25 - 0.75µm diameter).Suspensions of these cells can be concentrated on the filters by passage through a syringe/filter holder unit which is commercially available. The filters can be removed and passedthrough the various fixatives and into the CPD. After drying, via CPD or air dried throughthe organo-silicon compounds described earlier (TMS or HMDS), the filters may be cut tofit on the stub surface (conversely, large stubs may be utilized) and glued and conductivecoated. As an alternate, bacteria can be attached to a polylysine coated cover slip which isthen passed through the various fixation, drying, mounting and coating steps. Since youwill mainly working with single cells, the various protocol durations can be reducedconsiderably.

Protozoa are considerably larger than bacterial cells and are contained in a variety of waysfor fixation. Many individuals have modified large and small BEEM capsules as microbialcontainment vessels. A hole is cut into the cap and fine mesh filter is put in place afterloading the cells. The loaded BEEM capsule is placed into a vial for passage through thevarious fixatives. Diffusion of fixation agents occur across this microbial barrier. Aconvenient choice of a diffusion barrier is Nitex mesh which is available in a number ofmesh sizes. For most protozoa, a 40µm mesh is adequate. Nitex mesh, also known asbolting cloth, is a nylon material which is purchased by the yard.

Preparation of Nitex Bags

In this lab, a 40µm mesh Nitex bag is fashioned to contain our protozoan cells.Approximately 2" squares of Nitex are cut out and folded in half. An old pair of forceps isused to hold the free edges of the folded cloth together with about 1.0mm of the edgesextending beyond the forceps. A bacterial transfer loop is heated in a flame and run alongthe tightly held edges of the Nitex cloth. The excess 1.0mm edge is cut off whilesimultaneously sealing the free edges of the cloth. The cloth now has two open ends, one ofwhich is sealed in the identical manner by rotating the cloth square 90˚ and holding one ofthe two open edges in the forceps. The edge is sealed and the bag now has one open endinto which the protozoa are loaded. Nitex bags should be prepared in advance of thefixation. In addition, a number of micropipettes should be made before the actual time of


fixation by pulling standard Pasteur pipettes in a flame. The micropipettes prevent thetransfer of large volumes of culture media and fixatives during the fix process.

Microbial Protocol

As mentioned earlier, due to the size of the typically single cells, the duration of fixationsteps is greatly reduced as compared with large tissue blocks. For protozoa, a Quick Fixmethod is employed which involves mixing buffered Glutaraldehyde and aqueous OsmiumTetroxide. From the chapter on TEM specimen fixation, you will remember thatglutaraldehyde and osmium tetroxide react to form an undesirable precipitate. It ispermissible to mix these two fixatives in this Quick Fix since the samples will be removed(approximately 15 min.) before the precipitate reaction has become a problem leading tothe formation of surface artifact. The Quick Fix is carried out in the wells of transparentglass blocks, under a stereomicroscope. The stereoscope is placed under and at the front ofa fume hood to reduce the chance of vapor contact with investigator eyes. Multiple wells ofa white porcelain plate are filled with buffer solution for transfer into after the quick fix.The initial solutions required are:

• 3% Glutaraldehyde (in 0.1M phosphate buffer, pH 6.8 - match to culture medium)• 0.1M phosphate buffer, pH 6.8 (for washing)• 4% aqueous OsO4 (purchased in sealed ampoules)

A good log phase (high density growth) protozoa culture is desirable. Using a 1.0mmserological pipette, 0.5ml of the culture is transferred to a microfuge tube and placed into amicrocentrifuge head. A tube of water is marked and placed in the head opposite theculture tube for the purpose of balance. The microfuge timer is advanced to provide a burstof speed but without engaging the timer to even 1min. duration (which may be too forcefuland could damage your cells at 18,000 x g). When the head has stopped, the culture tube isremoved and the cells should be concentrated at the bottom. The Quick Fix solution shouldbe mixed just before the culture tube is spun down. It is prepared by mixing 2 drops of thebuffered 3% Glutaraldehyde to 1 drop of the aqueous 4% OsO4 in the glass depression wellwhich sits on the stage of a stereomicroscope, under the hood. If a fume hood isunavailable, a fan should blow across the microscope stage to prevent fumes from comingup into the face of the investigator. Do not contaminate solutions by using the samepipette for mixing!

Once the Quick Fix is mixed, a micropipette bulb is depressed and inserted gently to thebottom of the culture tube. The bulb is released and a large number of cells should enterthe micropipette. The contents of the micropipette are “shot” into the quick fix and allowedto remain for 15 minutes and no longer! The use of white photographic mounting boardunder the glass well block will enable you to easily observe the fixed and darkening cellsunder the stereoscope. As they fix, the cells will concentrate at the center bottom of thewell as they become impregnated with the high density heavy metal, osmium. As cells inthe first well fix, other wells in the block may be prepared as the number of samples yourequire dictates.


Once 15 minutes has elapsed, a fresh, clean micropipette is used to transfer fixed cells tothe buffer solution in the wells of a white porcelain plate (white is ideal since dark fixedcells will stand out in contrast against the white porcelain background). Once again,pressure is held on the micropipette bulb as the tip is advanced into the bottom of thefixation well. You must view this under the stereoscope so as to carefully guide the tip tothe area of concentrated cells. You must attempt to avoid transferring excessivefixation mixture to the buffer and prevent a precipitation reaction! With somepractice, you will readily suck up the fixed cells from the bottom of the well with aminimum of fixative. Depending on the number of cells initially transferred into thefixation well, you may perform a number of transfers out of that well and into a number ofbuffer wells using the same micropipette. At this point you should concentrate on gettingall of your fixed cell out of the quick fix mixture wells and into the buffer wells. The bufferwell plate can now be taken out of the hood to the lab tabletop and placed on the stage ofanother stereoscope.

Specimen vials are filled with buffer solution and a previously prepared Nitex bag isinserted into the vial and buffer solution so that the open end sticks slightly out of thevial. A slipknot lasso of white (no pigments which may come out in the EtOH or freonseries) sewing thread is looped over the open end of the Nitex bag. A new, cleanmicropipette is used to transfer at least 20 to 40 cells into the open Nitex bag. The cellsare shot down into the lower portion of the bag submerged in the buffer solution to preventpremature drying down and surface tension collapse. The lasso thread is tightened inorder to seal the opening and the remainder of the typical soft tissue protocol continueswith a reduction in the duration of each step (see below). To prevent damage by pipettes tothe Nitex bags and escape of its cellular contents, solutions are decanted off by pouringthem out. New solutions are pipetted in from above the bags. It is unlikely that the cellswill dry down between protocol steps since the bags retain adequate moisture.

Once through the intermediate fluid series, the bags are pulled out of the vials and laidinto the freon TF/113 filled channels of the CPD boat. The wire mesh covers are placedover each channel for protection and the CPD run progresses as usual. While the CPD runoccurs, clean stubs can be prepared with glue transfer tab adhesive coatings. Uponremoval from the CPD boat, the cells within the Nitex bags are dry and very delicate.Holding the bag upright (thread sealed end UP), the bag is lightly tapped on the side toconcentrate cells at the bottom. The thread is pulled off or the top of the bag is cut off. Atthe now open end, an adhesive prepared stub is inverted and placed at the opening of thebag. The bag is inverted and lightly tapped again. Dry cells should sprinkle down on theadhesive surface and stick. This can be confirmed with a stereo microscope view of thesurface of the stub. The stub can now be conductive coated as usual (consider this arelatively flat surface and coat for 30 sec.). In addition to the above “sprinkling” procedure,some cells may be trapped in the Nitex mesh. Once again, this can be observed under thestereoscope. In this case, the Nitex mesh can be opened and glued directly to an adhesivecoated stub. The excess Nitex can be trimmed at the edge of the stub and the stub can besputter coated. It should be noted that there are many variations to the above microbialtechniques in the literature. These Nitex bags have proven very successful in this lab.


In addition, we are usually not concerned about cell orientation on the stub surface. Forsome investigations, this orientation is critical. Once on the surface of a stub, cells may berepositioned using a microneedle which is prepared by pulling a capillary tube in a flameand allowing the end to seal. It is best if the cells do not contact adhesive prior torepositioning by keeping them in the Nitex mesh as opposed to sprinkling them outrandomly onto an adhesive coated surface. The microneedle may be placed into a glue suchas Pliobond so that the cells adhere to the needle for specific orientation and finalplacement (dorsal vs. ventral, etc.).

The entire microbial protocol is listed below and a blank schedule sheet is provided on thefollowing page:

• Quick Fix - 15 min.• Buffer Wash - 2 X 5-10 min.• 30%, 50%, 70%, 95% EtOH - 5 min. each• 100% EtOH - 2 X 5 min.• 30%, 50%, 70%, 95% Freon TF/113 - 5 min. each• 100% Freon TF/113 - 2 X 5 min.• Critical Point Dry (or alternative)• Adhesive Mount on Stubs• Sputter Coat with Gold (30 sec. @ 50mT and 45mA)• Store in Desiccator

Microbes make very interesting and rewarding samples for examination under the SEMwith preparation of living samples being conducted in less than half the time required forbulk tissue samples.

✥ Correlative SEM to TEM Sample Preparation

It is often desirable to correlate images generated by a variety of instrumentation, such asLM, SEM and TEM, since their resolution ranges overlap. Sample blocks prepared for theSEM can be taken and prepared for conventional TEM. It is important that the soft tissueblocks be small (no larger than 1.0mm3) at the outset of SEM preparation for reasons offixative penetration. Once a sample has been viewed and photographed using the SEM, itcan be gently removed using acetone (Pliobond glue is readily dissolved using acetone andis a good SEM adhesive choice if one is contemplating this procedure). The tissue blocksare transferred to propylene oxide (3 changes for 10 minutes each) and embedded in epoxyresin using conventional techniques (see Unit 1).


Microbial Fixation Schedule WorksheetSEM Microbial Protocol - Immersion fixation of Single Cells



Quick Fix (2 parts 3% Glutaraldehyde in buffer to 1 part 4% aq. OsO4)

Buffer Wash

Buffer Wash

30% Ethanol 50% Ethanol 70% Ethanol 95% Ethanol 100% Ethanol (fill vials completely) 100% Ethanol (fill vials completely)

30% Freon TF/113 50% Freon TF/113 70% Freon TF/113 95% Freon TF/113 100% Freon TF/113 100% Freon TF/113

Critical Point Dry - in LCO2


Sputter Coat (Gold) - 50mT, 45mA


Unit 3 - Black & White Photographic Principles inElectron Optics

The black and white electron photomicrograph is the final technical product of the electronmicroscopist. Since the TEM and/or SEM image disappears when the filament isdeactivated, it becomes a necessity to capture a permanent image for the purpose ofdetailed analysis. Silver-based black & white photography has satisfied this requirementfor many years. Even though many EM labs have converted to digital image capture,storage and analysis/manipulation through the use of personal computers (PC’s), mostelectron microscopists still rely on photographic techniques in the course of their studies.

From earlier discussions on electron optical principles you should recall that in order toachieve the highest possible resolution, voltage stabilization circuitry is employed whichresults in an electron source with a single constant wavelength. This monochromaticsource reduces the effect of chromatic aberration which would limit resolving power. Theconstant electron wavelength means that “color” electron images are impossible (unlessthey are pseudo-colored using a computer or hand colored - Dr. Martin’s dyes yieldexcellent results in hand coloring of EM images when applied directly on photographicprints, especially SEM).

The quality of the final photomicrograph is dependent on all of the tedious steps thatpreceded its production. An individual experienced in EM techniques need only look at amicrograph to determine flaws in the technique of the individual, whether the flawsresulted from poor tissue fixation, tissue embedment (TEM), ultrathin sectioning (TEM),post-staining for TEM/conductive coating for SEM, EM alignment and operation or poorphotographic technique. Careful attention to detail at each stage in the process willusually produce quality results. It should be noted that the resultant photomicrograph willtypically show more detail that the TEM or SEM viewing screen image. This is due to thefine-grained characteristics of most EM films in comparison to course-grained TEMfluorescent view screens and low resolution, 625 line, SEM monitors/CRT’s.

Black & white photography involves a two step process:

1. Negative production (exposure and processing)2. Enlargement Printing (exposure and processing of photographic paper using a negative

in an enlarger)

UNIT 3 – BLACK & WHITE PHOTOGRAPHIC PRINCIPLES IN EM 85

Chapter 8 - Film and Paper Composition

Modern films are composed of four (4) important layers (fig. 13). The upper layer is aprotective anti-scratch layer which is usually dull/non-reflective in appearance. Below thisis the most vital layer to the process of photography, the emulsion layer. In this layer,silver halide (such as silver bromide) crystals/grains are suspended in a gelatin matrixsome 25.0µm thick. In this layer, the photochemical reactions required to produce apermanent, high resolution image take place. Under the emulsion layer is a support layercommonly composed of cellulose acetate (commercially known as “estar”). The final layer isknown as the anti-halation layer which prevents photon/electron backscatter to theemulsion layer. This layer is shiny in appearance, therefore, if you examine a sheet of filmyou should notice a dull/emulsion side and a shiny side. Most manufacturers (Kodak) willcut a small notch in the film to allow one to determine the emulsion side of the film evenin total darkness - the emulsion side is up when the film is held with the notch in theupper right corner (fig. 14). The emulsion side of photographic paper is easy to determinesince it is highly glossy under the bright yellow or amber safelight used in printing.

Fig. 13

Fig. 14

anti-scratch - dull side

Emulsion (silver bromide/gelatin)

Support/Base (cellulose acetate)

anti-halation - shiny side

Film Composition:

Film Notching Guide:

emulsion side is up


✥ Emulsion

The effect of electrons is similar to that of photons on light sensitive emulsions. As statedearlier, the emulsion layer is the active site of the photographic process wherein achemical reaction occurs. The emulsion layer is approximately 25.0µm thick and iscomposed of usually uniformly sized silver halide (such as silver bromide - Ag+Br-) crystalssuspended in a gelatin matrix. Depending on the film, the crystals can range in size from0.1 to 10µm in diameter. This crystal diameter relates to the film speed as shall be seen.

For film, this emulsion layer is coated on a cellulose acetate support or base. For paper,the emulsion is coated on cellulose-based paper (Kodak Kodabromide paper) or coated on aresin (Kodak RC paper).

When an energy source such as electrons or photons encounter a crystal, the crystal isdisturbed/rearranged in various regions. The crystal is said to be in an activated state,however, an actual image cannot be seen at this point. A latent image exists whichtheoretically can persist for years. This latent image must be converted to an actual imagethrough processing of the film or paper. The chemical reaction that occurs when electrons/photons encounter a crystal is reduction, the gain of electrons.

Relative to a gain of electrons, the reduction reaction can be summarized as follows:

Ag+ + e- ➙ Ag˚

In this case, silver ions are reduced to silver atoms. The main difference between electronversus photon effects on the emulsion is that electrons are 100% quantum efficient. Thismeans that a single electron is capable of activating a single silver halide crystal whereas,it takes 10 to 100 photons to activate a single crystal. Therefore, no true film speed existswith respect to electron effects on light sensitive emulsions. Since this is the case, theelectron microscopist can take advantage by using very fine-grained films for the highestresolution. This is true only when electrons bombard the film to form the image as in thecase of the TEM. In SEM, photons from a high resolution photo-CRT are activating theemulsion. It should be indicated that a single silver halide/bromide crystal is composed ofa large number of individual ionic bonded Ag+Br- molecules. During the reduction reaction,the crystalline lattice is rearranged yielding the latent image.

✥ Film Speed (ISO/ASA)

The film speed simply refers to the diameter of the silver halide crystals suspended in theemulsion layer. Fast films (400, 1000) have large crystal diameters. These large crystalsare more likely to encounter and react with photons under conditions of low source(photon) density. If you have limited light conditions (indoors without a flash) you willneed a fast film in order to record an image. The resultant enlargement prints are verygrainy and of low resolution due to the large crystal size. Slow films (25,100) require anadequate photon concentration since they are very fine-grained. The fine-grained films


result in high resolution prints even at high magnifications.

Since electrons are 100% quantum efficient relative to the silver crystals, we can takeadvantage of the slow, fine-grained, blue-sensitive films available, such as Kodak ElectronImage Film 4489 with an estar base and an ASA of 15. This 3.5" by 4.25" sheet filmpermits excellent enlargements of high resolution. As it is blue-sensitive, the same safelight used in enlargement printing, yellow or amber, can be used when handling this film.

Light sensitive emulsions, even when used to capture electron generated images, aretypically sensitive to certain wavelengths/colors of the visible spectrum. Safelights can beused to take advantage of this fact and allow the individual to handle films and papersunder some lighting conditions. When choosing the appropriate safelight filter, thewavelength selected must be below the threshold energy necessary to activate the silverhalide crystals of the emulsion. If the film is blue-sensitive, a yellow or amber OA or OCsafelight filter is appropriate. If the film is yellow-sensitive, a yellow safelight filter wouldprovide the necessary energy to activate the emulsion. In this case, a red filter could beused. Bear in mind that along with the appropriate filter, a low 10-15watt bulb isnecessary at a minimum distance of 4 to 5 feet.

✥ Supports (Base)

Film

In the past, emulsions were coated onto breakable glass plates. The advantage of glass isthat it contains no water and requires no desiccation prior to insertion into the TEM. Thisis not important in the SEM since images are taken off the high resolution recording CRTat atmospheric pressure. The modern support used for film is cellulose acetate or “estar”.Although the estar plates are easier to handle and not subject to breakage, they must behandled carefully to avoid dust, fingerprints and scratches. The cellulose acetate containswater and must be desiccated prior to insertion in the TEM. Most modern TEM’s have aplate dryer which is evacuated by a rotary pump.

Paper

Paper for enlargement printing is manufactured by coating an emulsion layer on eithercellulose paper (Kodak Kodabromide) or resin (resin-coated or RC paper). Since theprocessing chemicals are absorbed by the cellulose paper, it requires longer processingtimes, especially the final wash in running water of up to 60 minutes. With RC paper, onlythe emulsion is affected resulting in shorter processing times. By example, the final washtime is reduced to 4 minutes. For drying, the RC paper should be treated for removal ofexcess water using a sponge or squeegee and allowed to air dry. By contrast, the cellulosepaper should be dried in a heated drum or plate dryer, allowing the print surface tocontact the shiny ferrotype surface for a high gloss final print.

Papers are designated by both letter and number combinations. The letters refer to the


type of print surface such as matte or glossy. For scientific publications, the glossy surfaceis most desirable and is noted by the letter “F”. The numbers (usually 1 through 5)indicate the paper grade or contrast level of the paper. Paper grades will be covered inmore detail under the topic of enlargement printing.

✥ Routine Photographic Films and Papers Used For TEM and SEM

Films

TEM: Kodak Electron Image Film 4489 (3.25 x 4", ASA=15) - OA or OC safelight.SEM: Kodak Commercial Film 4127 (4 x 5", ASA=50) - A1 (red) safelight.

Polaroid Type 55 P/N Film (4 x 5" positive and negative, ASA=50) - no safelightrequired.

Paper

For use in the enlargement printing of any of the negatives listed above:

Kodabromide F, 1-5 (fiber based, single or double weight) - OA or OC safelightKodak Polycontrast F - RC (resin coated, requires a Polycontrast Filter Kit) - OA or OCsafelight


Chapter 9 - Processing (Films and Papers)

Processing involves a series of steps that will transform the latent image, produced viaexposure of the film or paper, into an actual image made up of dark, light, and gray(halftones) regions, namely, contrast. The image will also be fixed and preserved on thefilm or paper by these procedures which include: Development, Stop Bath, Fixation,Washing and Drying.

The ideal way to process negatives involves loading the exposed film into the appropriatesized open frames/holders and pass them through the required solutions which are storedin tanks with floating lids (fig. 15). Papers are usually processed in various sized trayswith washing carried out in a unit which has an attachment for circulating water, such asa large tub which houses a rotating drum (fig. 16). It is important that the films andpapers be agitated throughout the entire process. Agitation reduces the chance that airbells (bubbles) on the surface will prevent necessary contact with the various processingsolutions.

Figs. 15 & 16 – Film & Print Processing Stations Illustrations

RunningWater Wash

20-30 min.

H C

Developer(D-19)

1:2

4 min.

Stop BathWater

1.5 min.

Rapid Fixerstraight

2-3 min.

Negative Processing Station - Tanks

NOTE: Times and dilutions indicated pertain to Kodak 4489 TEM Film

H C

Developer(Dektol)

1:2

1-2 min.

Stop Bathwith

Indicator

5-30 sec.

Rapid Fixer1:1

2 min.

RunningWater Wash

4 min.

Print Processing Station - Trays

NOTE: Times and dilutions indicated pertain to Kodak RC Paper


✥ Development

Development is the most critical step in processing. Under-development produces animage of poor, “muddy” contrast and low grain density (the image will appear too light).Conversely, over-development will result in “fogging” and produce a dark image (highgrain density) of poor contrast.

In development, the latent image on the film or print is essentially soaked in an “electronbath”, specifically, a reducing agent which will give up electrons to the silver atoms (whichwere initially reduced from silver ions during exposure to the source electrons or photons).During this “electron soak”, reduction of silver atoms to black metallic silver occurs. Thelatent image is transformed from activated crystals to actual black grains which yieldcontrast and a true, observable image. This reduction to black metallic silver begins at a“nucleus” in the activated crystal and spreads. Evidence of this can be seen byexamination of these silver grains under the TEM as is done in TEM autoradiography. AtTEM resolution, the grains appear elongated and worm-like versus their circular, dot-likeappearance under the light microscope. In order to prevent inactivated crystals from beingreduced, leading to fogging, development must proceed under very specific times andtemperatures.

The main components of a developing solution are as follows:Hydroquinone - the reducing agent. It requires an alkaline environment

(pH 10-11) in order to donate electrons.Borax or Sodium metaborate - provides the required alkaline pH 10-11.Sodium sulfite - preservative, prevents air oxidation of developer, lengthens shelf

life.Potassium bromide - slows development, serves as a competitor of the reduction

reaction, allows for control over the development process.

Typical developers include D-72 (Dektol) which is used primarily for papers and D-19, ahigh contrast developer used for films in scientific applications and x-rays. Thesedevelopers are available in powdered form and are mixed into one gallon of water at 90-120˚F. A good working solution has a characteristic odor, no precipitate/sediment, is clearwith a slight yellowish color, and is slippery/slimy to the touch due to the alkaline pH. Astock solution in a tightly capped bottle has a shelf life exceeding one month. Mostworking developer solutions are diluted from the initial stock solution. Development timesand temperatures must be carefully monitored to ensure that only activated silver grainsdevelop.

Development parameters for common TEM/SEM films and papers are as follows:

Film Developer Dilution Time TemperatureTEM Film 4489 D-19 1:2 4 min. 20˚CSEM Film 4127 D-19 1:1 4 min. 20˚CSEM Polaroid 55 Instant processing - no developer required


PaperKodabromide Dektol 1:2 1-2 min. 20˚CPolycontrast RC Dektol 1:2 1-2 min. 20˚C

One should be careful not to pull a developing print out of the solution too early. Under anOA/OC safelight, the image appears much darker than under normal white lightconditions. Since you actually observe the image develop, the tendency for the novice is tomove the print out of the developer before the recommended times as listed above. If theprint is too dark after the minimum time has elapsed, adjustments to exposure time or f-stop will be necessary.

✥ Stop Bath

As indicated by its title, the stop bath stops the development process. Typically, the stopbath is a 1% acetic acid solution which contains the pH indicator, Bromthymol Blue.Kodak markets this solution as Indicator Stop Bath; it is mixed with water in a typicaldilution of 1:62 (16ml per liter of water). The purpose of the acetic acid is to neutralize thealkaline development solution (unless the pH is 10-11, hydroquinone will not act as areducing agent). The bromthymol blue is used to gauge the acid-base neutralizing capacityof the stop bath. At a pH below 7.0, this indicator appears yellow and above pH 7.0, itappears blue. Under the OA/OC safelight, the solution will darken to indicate that it is nolonger effective in stopping development and should be replenished.

If it is changed often, water can also serve as an effective stop bath and is recommendedfor the TEM films (Kodak 4489) since the production of a mottled appearance on the filmshas been reported with the use of indicator stop bath. The water in the film developmenttank should be changed after each group of films (a quantity 8 to 10, in their holders) hasbeen passed through. Kodak 4127 film can make use of the indicator stop bath, however, ifthe same tanks are used for SEM and TEM processing, water will suffice. For paper, theuse of indicator stop bath mixed into a development tray of water is recommended.

Stop Bath parameters for common TEM/SEM films and papers are as follows:

Film Stop Bath Time TemperatureTEM Film 4489 Water 1.5min. 20˚CSEM Film 4127 Water or Indicator 30 sec. 20˚CSEM Polaroid 55 Instant processing - no stop bath required

PaperKodabromide Indicator Stop Bath 30 sec. 20˚CPolycontrast RC Indicator Stop Bath 30 sec. 20˚C

Almost continuous agitation in the stop bath is recommended due to the short timeduration required.


✥ Fixer

Photographic fixer, not to be confused with the fixation of biological samples, serves threeimportant purposes. Firstly, and most importantly, it contains a component which releasesinactive silver halide from the emulsion layer. This component, commonly known asHypo, is actually Sodium or Ammonium thiosulfate (or thiocyanate) which reacts with theinactive, ionic bonded silver halide to yield soluble silver thiosulfate/thiocyanate. In otherwords, the hypo allows for the inactive crystals remaining in the emulsion layer to dissolveinto the fixer solution. In large photographic processing labs, the fixer may be collectedand sold back to the film manufacturer who extracts the silver halide to be used in themanufacture of new film. The second ingredient is potassium alum which hardens thegelatin matrix of the emulsion to protect and preserve the image. This component is oftenreferred to as the hardener. The final ingredient of fixer is an acid, usually acetic orsulfuric, which aids in the hardening process and also can neutralize any developer carriedover from the stop bath.

Fixer is sold in powdered form (Kodak Fixer) or liquid form (Kodak Rapid Fixer) whichuses the ammonium thiosulfate based hypo. When preparing the stock solution, watertemperature is critical. If not adhered to, the final solution will appear cloudy and may notclear upon standing. The stock solution is usually prepared in one gallon capacity bottlesand used straight for films, and in the case of rapid fixer, diluted 1:1 for papers. Toprepare a gallon of stock rapid fixer for films, approximately one-half gallon of water at16-27˚C is added to the bottle. The entire contents of solution A is slowly poured into thewater, followed by solution B (caution: strong acid) added in small volumes followed byagitation. Water is finally added to bring the total volume to one gallon, followed bymixing.

The final solution has a pungent odor (acidic), is clear and colorless and has a long shelflife (up to three months or more). To check the effectiveness of the fixer solution,hypocheck, a 4% potassium iodide solution can be purchased or prepared by mixing 4.0gof KI into 96ml of distilled water and dispensing into dropper bottles. To test the fixer, twodrops of the hypocheck are added to the fixer solution. If a milky white precipitateappears, the solution is exhausted and should be discarded.

During fixation, materials should be agitated and the normal white room lights can berestored half way through the process (i.e. if a 4 minute fix is called for, lights can beturned on after 2 minutes).For Polaroid negatives (Type 55 P/N), the company recommends the use of a sodiumsulfite solution in order to clear the film, for 4 minutes duration. Straight fixer can also beused with satisfactory results.


Fixation parameters for common TEM/SEM films and papers are as follows:

Film Fixation Dilution Time TemperatureTEM Film 4489 Fixer straight 8 min. 20˚CTEM Film 4489 Rapid Fixer straight 2-3 min. 20˚CSEM Film 4127 Fixer straight 8 min. 20˚CSEM Film 4127 RapidFixer straight 3-4 min. 20˚C

SEM Polaroid 55 Fixer straight 4 min. 20˚C

PaperKodabromide Fixer straight 8min. 20˚CKodabromide Rapid Fixer 1:1 8min. 20˚CPolycontrast RC Rapid Fixer 1:1 2 min. 20˚C

✥ Washing

The purpose of washing the processed photographic materials in water is to remove any ofthe processing chemicals from these materials. If the chemicals are not washed off and outof films and papers, especially the fixer, these materials will brown with the passage oftime. It is important to adhere to the specific times and temperatures for washing,especially for fiber-based papers. Over-washing a fiber-based paper may result in creasingand possible tears as the paper is dried. Colder temperatures will require longer washtimes with the ideal temperature at 20˚C. Washing should always be done in a circulatingwater bath with constantly running water. Films can be left in overflowing tanks andpapers can be placed in specially designed paper washers, such as large tub washers withrotating drums and upper and lower drains.

Washing parameters for common TEM/SEM films and papers are as follows:

Film Wash Time TemperatureTEM Film 4489 Water 20-30 min. 20˚CSEM Film 4127 Water 20-30 min. 20˚CSEM Polaroid 55 Water 10 min. 20˚C

PaperKodabromide Water 30-60 min. 20˚CPolycontrast RC Water 4 min. 20˚C


✥ Drying

Drying of photographic materials, especially negatives, should be done in as dust-free anenvironment as possible. After washing, films should be dipped into a 1:200 solution ofPhotoflo (Kodak), which is a wetting agent to promote even drying and the absence ofwater spots on the negatives. Films are then hung on some type of “clothesline” to air dry.After films are dry, they should be protected in glassine envelopes.

Kodabromide, fiber-based papers, are dried in a heated flat plate or drum dryer with ashiny ferrotype surface. With the print face-up, a roller squeegee is used to remove excesswash water. The print is then positioned so that the printed surface contacts the hotferrotype surface which yields the desired glossy surface (with F type papers). The paper isrolled into the drum dryer with typically a canvas overlay. In the plate dryer, a canvascovering is stretched over the back of the print(s). After about five minutes, the drum canbe unrolled (or the canvas cover pulled back) and the prints will simply separatethemselves from the ferrotype surface. The ferrotype should be routinely be cleaned withthe appropriate ferrotype polish (available at photographic supply stores).

Resin-coated (RC) papers are simply wiped with a soft photographic sponge or squeegeeand allowed to air dry with F papers yielding a glossy surface.


Chapter 10 - Negative Handling and Exposure (TEM & SEM)

In general, unexposed negatives (film) for TEM and SEM must be loaded into someappropriate light-tight carriers for insertion into the “camera” of the microscopes. Asdiscussed earlier, films are to be handled carefully under the appropriate safelightconditions. The film sheets should be held on their edges to avoid the transfer of oilycontaminants from the skin and the resultant area of poor exposure and processing. Thefollowing section will describe the loading and exposure of films for the TEM and SEMgenerally, and the Hitachi HS-8 TEM and the Hitachi S-2400 SEM, specifically.

✥ TEM - Hitachi HS-8

In the case of the TEM, films must be loaded directly into the vacuum environment of themicroscope, usually in a camera chamber located below the viewing screen. When thescreen is pulled out of the way, the imaging electrons will now contact the photographicemulsion and expose it, producing a latent image. Water-containing, estar based filmsmust be desiccated prior to loading and insertion into the TEM camera chamber.Otherwise, you can expect lengthy pump-down times and contamination/corrosion ofinternal TEM components. As stated earlier, modern TEM’s will usually come with a platedryer. Films should be dried at low vacuum for at least one hour prior to loading.

The modern TEM will usually have a light-tight storage/loading box (for storage ofmultiple unexposed film plates in the microscope) and a light-tight receiver box (forcollection of exposed plates) that will be placed into the TEM camera chamber. Withinthese boxes, the film plates are held, often in flat metal plate holders. For the Hitachi HS-8, 18 film plates can be loaded into a single storage/load box. A single sheet of film,emulsion side up (notch in the upper right corner), is placed into the metal holder and arectangular metal frame is added to keep it in place. It is important that the flat sideof the frame be placed down against the film and the beveled side face up –otherwise, the film holder might get stuck in the motorized camera mechanism.The lower edge of the frame is pushed into the spring arrangement at the base of themetal holder, while the upper edge of the frame is guided and held in the two, L-shaped,upper catches. The loaded plate holders are then stacked into the storage/load box, whoseentire front sliding door is removed, with the plate holder central notch pointing out. Onceagain, the box holds 18 plates at the maximum. Do not overfill the storage box! Oncefull, the storage box can be put into the desiccator/plate dryer for a minimum of one hourat low vacuum.

A desiccated storage box can then be transferred into the TEM camera chamber. In orderto do this, the TEM must be on and at high vacuum. The camera airlock must be closedand air admitted into the camera chamber through the camera door. The cover for the loadbox site is lifted straight up and off. If still in place, the old storage box is removed fromthe TEM and the new box put in its place. The arrow on the front door of the box andthe “F” (for front) must face the front of the TEM upon loading into the TEMcamera chamber. When in place, the front and rear doors of the box are slightly raised


so that the single lowest plate holder can be advanced forward under the TEM view screenby the motorized plate drive. Before placement of the storage box into the TEM, theblack blanking plate should be removed since they have become stuck in ourparticular scope in the past - this necessitates performing the insertion processunder safelight or dark conditions! A full receiver box should be removed and replacedwith an empty one. These receiver boxes can be found behind the front camera door. Theyhave a front handle and a hinged top lid which should be opened only under safelightconditions, when you are ready for processing. When the two camera chamber doors arereplaced, the chamber is rough pumped using the rotary pump (CAMERA button),followed by deselecting the CAMERA button (usually for PLATE) and opening thecamera airlock.

✥ HS-8 Camera System and Film Exposure

The camera system and film exposure mechanism of the Hitachi HS-8, as for other modernTEM’s, is essentially automatic. As the TEM operator, you must ensure a number ofconditions are met prior to activating the automated camera system for the best possibleimage capture. For example, if your image is under or over focused, the auto camerasystem will provide an excellent exposure of an out of focus image. This brief section willprovide a step by step procedure for capturing a properly exposed image of high resolutionand optimal contrast.

Initially, it is important to understand the basic workings of the TEM camera system.Within the lower region of the TEM column (observable through the small, lower circularviewport of the HS-8) four CdS photocells are located in the peripheral electron beampath. When energized, these photocells will begin to collect electrons (or photons) up to acertain sensitivity set point. At this set point, a metallic shutter mechanism is set inmotion which effectively blocks the electron beam from contacting the film emulsion. Theexposed film can be moved into a light-tight receiver box and later removed for processing.All of these events will occur in the high vacuum of the TEM column. As stated earlier,films for TEM must be desiccated prior to their insertion into the microscope.

The actual procedure of high quality image capture for the HS-8 follows:

1. Scan your grid at low magnification (1,000-2,100 X) and locate a section and area ofinterest. You will need to magnify, center, adjust brightness and focus in order to locate anarea worthy of an exposure. Avoid sections and areas containing obvious contaminationand sectioning artifacts.

2. Determine the magnification you will be exposing the film at. Important: if youchange the magnification you will have to perform all of the steps which followfor the new mag setting!

3. Focus the image using high brightness and the coarse and fine objective lens focuscontrols. The screen should be tilted and the 10X binoculars utilized for this process - the


binocs should be focused on the screen crosshair. If a small hole exists in the vicinity of thearea of interest, it is ideal as a focusing aid. The presence of interference fringes inside oroutside the hole indicates a condition of underfocus or overfocus, respectively. Note: tothe human eye, the underfocus image appears to be in best focus - this is thepoint of maximum contrast/granularity, also known as defocus contrast. Thehuman eye responds to the high contrast as the most desirable image. Truefocus is achieved when no interference fringe is observed in conjunction withthe hole - although not as high in contrast, true focus yields the highestresolution.

4. Frame the area of interest using the stage (X,Y) controls - note the small centralframing box on the main view screen. Note: the view screen must be in its flat, notangled, orientation for this framing process since the film will lie flat beneaththe screen.

5. Activate the Exposure Meter by depressing the button in the right auxiliary drawer.In order for the camera and auto exposure mechanism to function properly, the cameraairlock must be opened - a small square status indicator will light, a box imprinted withthe #0, on the left main panel.

6. Using both the Brightness knob (condenser lens current) and the electric deflection coil -Bright X, Y, spread out the beam to the screen periphery. This step is critical since thephotocells are located in a peripheral position. If the beam intensity is central, thephotocells will not be contacted directly and begin to collect the electron energy necessaryto close the shutter. An overexposure will result. The photocells must also be evenlyilluminated for optimum performance of the automated system. This is accomplishedusing the Bright X,Y in conjunction with the Brightness control.

7. Under “normal” conditions, the photocell sensitivity knob (the large black knob on theright main panel - labelled Exposure Meter) should be set to #5. By normal, I mean thatnone of the photocells are blocked by a grid crossbar(s) - this situation would cause thethree exposed photocells to do the work of four leading to an overexposure as the film iscontacted by the electron beam for an excessive time period. In an alternative situation, asection may be slightly torn away from a grid bar edge and directly over a photocell. Thetorn area will allow maximum electrons to contact this photocell and close the shutterprematurely - the result is an underexposure. Under these abnormal conditions, thephotocell sensitivity knob would have to be adjusted from the #5 position. As you go up to#6, #7, the sensitivity decreases for exposures taken with an intruding gridbar. Thesensitivity increases as you go down for exposures taken with tears in the resin.

8. At this point, an automatic exposure could be attempted, however, you may be wastinga tremendous amount of film if you do not perform this simple exposure time check beforeloading a piece of film under the view screen. To test the exposure time, lift the chromeplated shutter lever to the right side of the view screen - lift steadily and completely back.As you pull the lever back, you will hear the click of the microswitch with sets the auto


exposure system electronics into motion. At the sound of the microswitch, begin to time inseconds until the shutter swings into position to block the beam (observable through thelower view port) and the red LED comes on (above the photocell sensitivity knob). Theideal exposure time at sensitivity #5 is between 2-3 seconds! If this time is notachieved, adjust the intensity with the Brightness knob and try again until you get twoidentical, consecutive results. Obviously, this test should be done without film under thescreen.

9. You are now ready to perform an actual film exposure. With the screen flat, press theblack FEED button on the right main panel. The motorized plate drive will feed one pieceof film from the load box and move it under the main view screen. The illuminated statusbox #0 goes dark and box #1 lights to indicate the new position of the film. At this point,pull back the shutter lever and time the exposure (2-3 seconds). Wait for the closure of theshutter and the red LED activation. After the exposure, lower the view screen flat bypushing the shutter lever fully forward. The FEED button is depressed again and theexposed film plate is advanced into the open receiver box (the #1 light goes dark and #0 isonce again illuminated).

Exposed TEM films can eventually be removed from the microscope by closing the cameraairlock - this action also closes the light-tight receiver box internally. The films can beprocessed as described earlier in this chapter.

✥ SEM - Hitachi S-2400

Since all photographic exposures in SEM come off of an external, high resolution (2,000line) CRT, there is no need to desiccate films. In order to use the Polaroid Type 55 P/Nfilm, a 4x5" Polaroid camera back is required. It is locked into the camera box of the SEMphoto-CRT with two sliding clips. The silver lever on the Polaroid back is swung into the“L” (load) position and the film is inserted carefully into the slot. It is pushed all the wayto the back until a positive “click” is heard. The film is pulled out until it stops. The film isthen exposed as described later. After exposure, the film is pushed all the way back in andthe silver lever swung to the “R” (release) position (which is where it should be normallyleft when not in use). The film is pulled all the way out of the camera back and allowed toautomatically process for the time recommended (20-25 sec. at room temperature for type55 film). The film is pulled apart at the tabs and the positive print removed and coated asnecessary. Negatives are detached at the perforation and cleared in fixer or sodium sulfitesolution, washed and air dried. Rollers of the Polaroid back should be regularlychecked and cleaned of processing chemicals, especially if streaks appear on theprints/negatives - cleaning is done using water on a non-linting cloth.

If Kodak sheet film, 4127, is used, it must be loaded into the light-tight, dual cassette filmholders. These two-sided holders use a sliding blind to expose a loaded sheet of film in thecamera box of the photo-CRT. The cassettes are loaded under red safelight conditionsmaking sure that the films (emulsion side up) are slid into the lower film channel and notthe upper blind channel. The blind must be properly slid into the slot in the hinged


endpiece and the metal lock put in place. When ready for an exposure, the loaded cassetteis placed into the camera box of the photo-CRT and locked into place with the Polaroidback side clips. The lower blind is pulled out to expose the film in the box. The automaticexposure for the SEM is conducted and the lower blind is slid fully back into place andlocked. The cassette is then flipped over (if you forget to do this, a double exposure willresult) and another exposure is prepared. The exposed films can then be carried into thedarkroom for processing. Dry negatives are then evaluated (for focus, quality) andenlargement printed.

The S-2400 has a video output jack (RS-170) which allow for the connection of externalmonitors, video-recorders and thermal video printers. The addition of a frame grabber andcomputer allows for digital image capture, archiving and analysis (using programs such asNIH Image - a public domain program available on the internet) as well. A thermal videoprint (12¢) is an excellent way to determine preliminary image quality, focus,astigmatism, etc. before committing to the more expensive films ($1.00-2.00). For the bestquality video prints you can either take them using the slowest viewing scan pass (settingof 3-4) or perform a photo pass (without film loaded in the camera box), allowing the passto complete and the image to “freeze” on the viewing CRT. The frozen photo image is of thehighest quality for these video prints. It should be understood that the video printer iscapturing images at the lower resolution of the viewing CRT (625 lines) and not the photo-CRT (2,000 lines). Image capture using a VCR should also be performed under conditionsof slowest or photo scan rates for the highest quality images.

✥ S-2400 Camera System and Film Exposure

As for the modern TEM, a modern SEM possesses an automated camera and exposuresystem. The SEM differs in that exposures are taken off of a high resolution CRT atatmospheric pressure. There is no need to desiccate films prior to their usage in the SEM.There are also a number of conditions which must be satisfied for high quality imageproduction with the SEM. The following is a step by step method for producing thesequality images:

1. Scan stub at low magnification in order to locate tissue blocks, then specific areas ofinterest. Once an area of interest, such as a kidney Glomerulus, is located, a finalmagnification must be decided upon (for example, 5,000X).

2. If possible, raise the magnification by a factor of 10X higher than the desiredmagnification (in the above example, raise the mag to approximately 50,000X). At thispoint, align the final aperture using the focus wobbler switch “APER ALIGN” in thesubpanel - ideally you should observe a central pulsing of the image indicative of properalignment (typically use TV scan rate A).

3. Stigmate (STIGMATOR X,Y) the image using a high contrast image (CONTRAST/BRIGHTNESS controls) and a REDUCED AREA/RAPID scan rate. The best images tostigmate on are spherical in nature. Reduce or eliminate image stretching. The


STIGMATOR MONITOR control in the subpanel can also aid in image astigmatismcorrection.

4. Once the image is stigmated, the magnification is reduced to the original desired value(5,000X) and the image is framed on the viewing CRT (its entire area is the frame box).

5. Using REDUCED AREA/RAPID scan, the image is critically focused at the chosen magusing the MANUAL FOCUS controls.

6. The final image BRIGHTNESS & CONTRAST is adjusted using either AUTO (ABC) orMANUAL features. If ABC is used, select the auto mode (LED will light) and depress theABC button. ABC will appear on the viewing CRT and the SEM will make an audiblesound when the auto function is completed. This can be performed at any scan rate,however, I prefer a slow scan of 3 or 4. In addition, the first attempt is rarely optimal.Always allow three to four attempts of pushing the ABC button for best results (twoidentical brightness/contrast results are desired). Although ABC saves time in the printdarkroom since all of your negatives will be of identical exposure characteristics, there aretimes when it just will not yield quality images. This is especially true when a singleobject, such as a Paramecium, stands out against the stub background. ABC will simplyaverage the areas of high and low contrast and brightness and a muddy, washed-outimage will result. In order to emphasize the dark background and the raised, bright object,a MANUAL brightness and contrast adjustment must be performed.

Initially, select the LINE ANALYSIS button (press ONCE) and use the manual brightnessand contrast controls to keep the moving waveform within the bounds of the centralbright, band-like area which exists horizontally across the viewing CRT. Now select scanrate #2 and adjust for the highest quality image in conjunction with the line analysis data(moving waveform should be predominantly within this central brighter area for optimumphotographic brightness and contrast).

7. Once brightness and contrast is adjusted, select slow scan #3 or #4 and view the finalimage at high resolution - a video print is ideal at this point to critique your image beforeusing the film.

8. In order to expose a piece of film (either Kodak sheet film or Polaroids), it must beloaded/slid into the light-tight camera box (which faces the high resolution photo-CRT)and locked into place with the side clips. The film is exposed, face down in the camera boxas described earlier.

9. The exposure speed is checked (usually set on #2 - 80 sec. scan pass) and the photoSTART button is depressed. A scan line will slowly move down the viewing CRT as it doeson the photo CRT - “painting” the image one line at a time on the piece of exposed film. Atthe end of the photo run, the SEM will make an audible sound and the final image willdigitally FREEZE on the viewing CRT (the image will remain even if the filament current


is turned off). To eliminate the frozen image, select ANY other scan rate (A/B, 1/2, etc.)Kodak films can now be transported to the darkroom for processing. Polaroids can be autoprocessed and the final print received in approximately 20-30 seconds. Type 55 P/N printswill need to be coated, while the negative will need to be cleared in a fixer or sodiumsulfite bath as described earlier. Polaroid Type 53 films are coaterless and do not yield anegative. Its 400 speed (ASA/ISO) will also require that you stop down the lens in thephoto-CRT camera box. The other SEM films discussed thus far have a speed of 50.


Chapter 11 - Enlargement Printing

Enlargement printing is the final step in the production of an electron micrograph. Theenlarger is used to expose a sheet of photographic paper to light which passes through anegative. Areas on the negative with high grain density (i.e. dark/black areas) willinterfere with the passage of light to the photographic paper. This corresponding area onthe photographic paper will receive little or no exposure and therefore appear white afterprocessing. Conversely, areas of low grain density on the negative will allow the passage oflight to the paper thus exposing it. Subsequent to processing, these areas will appear dark/black. The result is a positive print or electron photomicrograph. Since the enlarger lenscan be positioned at various heights above the photographic paper (usually held in aneasel), a large range of magnifications/enlargements (or even reductions) of the originalnegative are possible.

Enlargers used for the printing of EM negatives must be able to accommodate largeformat negatives up to 4" X 5". In terms of enlarger design, the main unit possesses a 110vlamp housing followed by a condenser lens assembly. A variable condenser assembly iscommon which must be adjusted depending on the size dimensions of the negative. Next,there must be some provision for the insertion of the negative holder which contains thenegative, below the condenser. Appropriate size negative holders are required whichtypically sandwich the negative between two metal plates. The negative should be loadedwith the emulsion/dull side down (shiny side up). If in doubt as to the dull vs. the shinyside of the negative, use the notching guide. Below the negative you will find the enlargerlens assembly with a provision for removing and changing it. The focal length of the lensshould be printed on it somewhere, along with the clickstop apertures or f-stops. Rotationof the f-stop ring will allow you to select different apertures. In deciding which lens focallength to use, measure the negative on the diagonal in millimeters. The enlarger lens focallength should be equal to or slightly larger than the negative diagonal measurement inmillimeters. For 35mm negatives, a 50mm lens is used. For both TEM and SEM negatives,a 135mm focal length lens is a good choice. In the main unit of the enlarger, you mightalso find a site for the insertion of filters (such as polycontrast filters). If this is notavailable on the enlarger, a filter kit and filter holder which attaches to the enlarger lensis available for purchase at photo suppliers. A solid support platform of wood or metal anda rack and pinion gear system (motor or hand driven) to move the main unit closer to oraway from the support base/platform completes the basic enlarger design. Additionaluseful equipment would include an easel (adjustable to a variety of paper sizes), a grainmagnifier for fine focussing, and an automatic timer for convenience in timing exposures.

✥ Photographic Paper Grades

Photographic paper is available in a variety of different surfaces/textures and contrastgrades. For scientific work, the glossy surface (designated “F”) is the standard. Papergrades, usually numbered 1 through 5, refer to the contrast level of the paper. Number 1paper is soft or low contrast and number 5 is hard or high contrast. The ideal negative can


be described as possessing black blacks, white whites and a range of grays or halftones.This ideal negative should be printed on #3 paper. If the negative is high in contrast, itshould be printed on low contrast, #2 or #1 paper to compensate. Similarly, a low contrast,muddy gray negative should be printed on a high contrast, #4 or #5 paper. These differentpaper grades can be purchased separately or a single paper, known as Polycontrast, with arange of paper grades is available. Polycontrast paper is designed with two emulsions; ahigh contrast purple sensitive emulsion and a low contrast yellow sensitive emulsion.These emulsions are selected for by the use of various purple and yellow shadedpolycontrast filters. The advantage is that only one type of paper is purchased and thepaper grade ranges are finer, with half-steps available (e.g. 4.5, 3.5, etc.).

✥ Process of Enlargement Printing

Firstly, the darkroom must be setup. Trays of Dektol (1:2), indicator stop bath, and rapidfixer (1:1) are prepared and OA/OC safelight(s) activated. All paper grades or Polycontrastpaper should be available in a light-tight paper safe. Wash water can be flowed into theprint washer (which ideally is located near the fixing bath for ease of transfer. The easelshould be set for the appropriate size paper (i.e. 8" X 10"). The variable condenser shouldbe set to the negative dimensions and the proper focal length lens inserted and adjusted tothe largest aperture (smallest f-stop).

The negative is carefully loaded into the negative holder emulsion side down. Dust andfine hairs can be removed using a camel hair blower brush. Be careful not to scratch thenegative or transfer fingerprints to its surface. The negative holder is inserted into theenlarger unit and the light for focusing is turned on (if a timer is used, it will have someprovision for energizing the light for focusing and then turning it off for automatic timedexposures). With the room lights off, the negative is focused on a sheet of white paper inthe easel using the enlarger focusing knob. In order to fill the frame of the easel, it may benecessary to raise or lower the enlarger lens. You may want a high magnification print ofa selected area, in which case, you will have to raise the enlarger and frame the area ofinterest by moving the easel. The level of magnification can be determined later either bymeasuring the same objects on the negative and print in millimeters and by dividing theprint object length by the negative object length or by placing a clear metric ruler in thenegative carrier and comparing it to a metric ruler on the easel.

The image must be framed and focused (preferably using the grain magnifier) with theaperture wide open to ensure the maximum available light. After framing and focusing,the lens is stopped down since the quality area of the lens is not at the periphery. Theenlarger light is turned off. At this point, a test print must be made to determine properexposure time and contrast level. One method is to expose one-sixth of a sheet ofphotographic paper using a rectangular cardboard cover. The enlarger light is turned onand the paper exposed for 10 seconds. The enlarger light is turned off and the second one-sixth of the paper is uncovered by sliding back the cardboard mask. This area is exposedfor 10 seconds, and so on, until the entire piece of paper is exposed for a final 10 seconds.Of course, the first exposed strip will have been cumulatively exposed for 60 sec., the next,


50 sec., and so forth. The other method is to use a Kodak Projection Print Scale whichis a pie wheel with wedges of increasing density. Each wedge is numbered in seconds ofexposure time. To use it, the Print Scale is placed on top of the test piece of photographicpaper and a 60 second exposure is done. The test print is processed, as discussed earlier inthe chapter, and each wedge judged for the optimal exposure time which can be readdirectly off the wedge. It should be noted that initial test prints should be done using #3paper grade unless you have experience judging negative contrast levels. The test printalso serves to determine whether the contrast level of the print is adequate or if a changeof paper grade is in order. Changes of paper grade require a modification in exposure timewhich will be covered later.

If the contrast of the test print is satisfactory, the correct exposure time must bedetermined. Exposure times between 15-25 seconds are ideal. Long exposure times mayintroduce vibration effects to your focus while short exposures lack control over consistentduplications. If necessary and if possible, the f-stop should be adjusted to allow for a finalexposure time of 15-25 seconds, based on the results of the test print. Once the properexposure time is determined, the timer is set and a fresh sheet of paper is loaded into theeasel (be careful that you have not moved the position of the easel). The paper is exposedfor the correct time followed by processing, washing and drying.

✥ Enlargement Printing Variables

Three common variables in enlargement printing are increases/decreases in magnification,contrast/paper grade and lens aperture diameter (f-stop).

When exposure time has been determined for a certain enlargement and the magnificationis then increased, the exposure time will also have to be increased since there is a greaterspread of the light. Less available light means having to increase exposure time for a printcomparable in quality to the lower magnification print (relative to grain density andcontrast). Reduction in magnification requires a reduction in exposure time. The followingcalculation provides an approximate change in exposure time when changingmagnification:

(M2 + 1)2

T2 = T1 ––––––––––(M1 + 1)2

where:T2 = the new exposure timeT1 = the old exposure timeM2 = the new magnificationM1 = the old magnification

Relative to changes in paper grade, an increase in paper grade, say from #3 to #4, willrequire an increase in exposure time. A decrease in paper grade will require a decrease in


exposure time. A calculation for approximating such changes in exposure time relative tochanges in paper grade can be made since each paper grade has a reference printingindex. The print index for each paper grade is listed below:

PAPER GRADE PRINT INDEX1 50002 32003 20004 12505 1000

The calculation based on these print indices follows:

I2 T2 = T1 –––––

I1

where:T2 = the new exposure timeT1 = the old exposure timeI2 = the new paper grade print indexI1 = the old paper grade print index

In terms of a change in lens aperture or f-stop, one must firstly understand that the higherthe f-number, the smaller the diameter of the lens opening or aperture. Therefore, the f8opening is larger than the next stop, f11. To be specific, the next stop up is one-half thediameter of the stop below it and will admit only one-half as much light. By example, anexposure of 60 sec. at f11 is equivalent to 30 sec. at f8 and a 15 sec. exposure at f5.6. A testprint indicating 60 sec. at f11 could be brought into ideal exposure time by opening thelens to f5.6 and exposing for 15 seconds. Conversely, a test print that indicates 5 sec atf5.6 could be stopped down to f11 and exposed for an equivalent 20 seconds.

✥ Printing Tricks

If attention to detail is given throughout all stages of EM sample preparation, microscopeoperation and photography, resorting to darkroom “tricks” will not be necessary. Thefollowing are intended to yield a more uniform and professional quality print.

When areas of a negative are extremely dark, the result on the print will be a stark whitearea, such as the unexposed paper appears. To provide some background grain density, aprocess known as intensification can be used. While in the developer solution, thesurface of the white areas are rubbed with a couple of fingers. Body heat causes theactivation and development of some crystals in the area.

Burning-In involves negative regions which are again too dense/dark which produces


washed out print areas. These areas can be burned-in by exposing the entire print for theideal exposure time (as determined by test print) and then adding some additionalexposure time to the excessively white print region while masking the rest of the printsurface. You can purchase cardboard masks for this purpose or you can simply use yourhands at differing distances from the lens to form a suitable mask. While the additionalexposure time elapses, it is imperative that you keep the mask or your hands moving inorder to prevent a darker outline of the burned-in area.

Dodging-Out is similar to burning-in except that some regions on the processed print areexcessively dense/dark due to a low grain density negative area. In this case, the idealprint exposure time is determined by test print along with the area of concern. During thecourse of the ideal exposure time, this low density negative region is masked at few secondintervals throughout the exposure. Do not forget to keep that mask moving using shakylateral motions.

manual of electron microscope

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