techniques of advanced light microscopy and their applications to morphological analysis of human...

Techniques of Advanced Light Microscopy and TheirApplications to Morphological Analysis of HumanExtra-Embryonic MembranesC.D. OCKLEFORD,* L.C. MONGAN, AND A.R.D. HUBBARDDepartment of Pre-Clinical Sciences, Maurice Shock Building, University of Leicester Medical School, Leicester, UK

KEY WORDS confocal microscopy; placenta; amnion; fetal membranes; immunocytochemistry;fluorescence microscopy

ABSTRACT The science of light microscopy has advanced dramatically in recent years throughthe introduction of new technology. A brief description of scanning light microscopes, laserillumination, the confocal principle, digital imaging, and image processing reveals a number oftheoretical advantages which are particularly useful in improving epifluorescence microscopeimages.Examples of results from several studies of human extra-embryonic membranes conducted in our

laboratory show how the application of these techniques has been used to describe structures suchas microtrabeculae and rivets for the first time, to map the microscopic distribution of a wide rangeof proteins, and to observe the activity of placental villi at the microscopic level in an environmen-tally controlled microscope stage.High-sensitivity detectors have permitted the ‘‘super-resolution’’ detection of structures smaller

than the theoretically calculated limits of light microscope resolution.Rendering images in false colour is demonstrably useful in detecting subtle variations in

fluorescence intensity at different intracellular sites and at different sites within tissues of fetalmembranes.Processing stacks of digital images using appropriate software allows the 3-D reconstruction of

suitably sized extra-embryonic membrane components. These digital images created from opticalsections through the tissue are obtained non-destructively, and the relationships in space of thecomponents are well preserved.Microsc. Res. Tech. 38:153–164, 1997. r 1997 Wiley-Liss, Inc.

INTRODUCTIONBecause the nature of advanced light microscopes is

so varied there is no unique event which can beidentified as an origin. Rather, the origins of advancedlight microscopy are to be found as a number oftechnical advances in unrelated fields such as comput-ing, laser optics, image processing, and informationtechnology. The realisation that these could be helpfulto microscopists was initially restricted to a few whowere interested in the development of microscopicaltechniques. However, as understanding has becomemore widespread among those applying microscopicmethods to biomedical problems, there is a clear appre-ciation that life-science research has gained greatlyfrom their introduction. This resulted in the followingjustifiable claim made by Inoue (1989) when describingone of these instruments, the confocal laser scanningmicroscope (CLSM): ‘‘Seldom has the introduction of anew instrument generated as instant an excitementamong biologists as the laser scanning confocal micro-scope.’’ In this article, we will review some of therelevant features of this and allied instruments andthen cite some examples from our recent published andpreviously unpublished research to demonstrate thevalue of the methods in research on extra-embryonicmembranes.

PROPERTIES OF ADVANCED LIGHTMICROSCOPES

Scanning Light MicroscopesThe alternative of scanned beam illumination sys-

tems to the conventional flood beam systems are wellrecognised by electron microscopists. On the otherhand, they have been available and relatively unex-ploited by light microscopists for most of the 44 yearssince Young and Roberts (1951) converted an oscillo-scope into the first scanning light microscope illumina-tion system.There is an advantage in the use of flood beam

instruments for increasing temporal resolution becauseall parts of the specimen under observation are viewedat the same time. However, by restricting the area ofthe specimen under illumination at any one time byscanning a very narrow pencil of light over it, theopportunity for ‘‘cross-fire’’ light scattered within onepart of the specimen to degrade the image of neighbour-ing parts is minimised. Thus, at the expense of speed of

Dr. L.C. Mongan is now at the MRC Toxicology Unit, Hodgkin Building,Lancaster Rd., Leicester LEI 9HN.*Correspondence to: Dr. Colin Ockleford, Advanced Light Microscope Facility,

Department of Pre-Clinical Sciences, Maurice Shock Building, University ofLeicester Medical School, University Road, Leicester LE1 9HN, UK.Received 15 March 1995; Accepted 4 September 1995

MICROSCOPY RESEARCH AND TECHNIQUE 38:153–164 (1997)

r 1997 WILEY-LISS, INC.

image acquisition resolution can be increased margin-ally. Indeed, the so-called x,y resolution (point resolu-tion in the plane orthogonal to the optical axis) of someof the currently manufactured instruments approachesthe theoretical maximum calculated byAbbe (1884).The confocal laser scanning microscopes in common

use are nearly all beam scanning devices. These employgalvanometer mirrors which might scan a specimencompletely once and produce a full screen image in 2sec. Repeating a line scan over the specimen at thesame depth (x,t scanning) allows sampling of repeat-times in themillisecond range, and some useful informa-tion regarding rapid physiological changes reported byion-sensitive vital dyes can be gleaned.Scanning a line at successively deeper planes in the

specimen (by advancing step-wise a motor-driven focuscontrol) and displaying these lines in sequential rankson a pixel display can give rise to a lateral view (an x,zscan) of the specimen remarkably rapidly.Increased scanning speeds are obtainable by the use

of acousto-optical modulator scanning systems (Suzukiand Hirokawa, 1986). These allow video-rate scanningbut are restricted in their field width compared withgalvanometer mirror scanning systems. One of theearliest of the confocal microscope systems to be made,the scanning tandem microscope (Egger and Petran,1967) is based on the multiple-beam arc-scanning illu-mination produced by a perforated spinning disc(Nipkow, 1884). This ‘‘real-time’’ system sacrifices illu-mination brightness and requires high-sensitivity detec-tors for optimum function.The balance between scan-speed and resolution can

be struck differently by using a slit beam rather than aspot of light to scan the specimen in less time. This typeof apparatus leads to a useful direct view (‘‘real-time’’)instrument, but with slightly lower resolution thanspot scanners provide.The greatest field-width and the highest resolution

scanning light microscopes are provided by axial illumi-nation in which the beam is fixed while the specimen isscanned on a moving stage (Brakenhoff et al., 1985).Using this approach, large specimens such as wholeembryos can be scanned. This process of stage scanningis the slowest of the scanning methods. The mechanismfor precise movement of the stage is also difficult andexpensive to produce.

Laser IlluminationThe availability of lasers which offer coherent beams

of intense monochromatic light with low divergencecharacteristics suitable for scanning purposes has beenone condition that has added impetus to the recentadvances. The use of monochromatic light eliminateschromatic aberration, and the high-intensity illumina-tion makes the instruments more useful as dwell timesare reduced and scanning speeds can be increased.Among lasers currently used for microscopic illumina-tion there are a variety which are in common use(Gratton and vandeVen, 1989), including Argon ion,Helium, Neon, Krypton Argon, RGB lasers, and UVlasers, which all have strong lines in different parts ofthe spectrum which can be applied to different imagingor experimental conditions. In conjunction with theappropriate optics, dual and three-channel illumina-tion systems allow an extended range of capabilities

(vide infra). There is a branch of photochemistry whichis now highly active designing fluorescent chemicalswith the appropriate absorption and emission character-istics for use with restricted wavelength illuminationsystems. Several of these have proven practically useful(Ockleford et al., 1981; Waggoner et al., 1989). Thepower of the laser illumination of these systems can beused for experiments investigating fluorescence recov-ery after photobleaching.

Confocal OpticsThe confocal principle is neat and simple (Minsky,

1957). Essentially, it is a form of aperture processing.Two pinholes (the confocal apertures) are placed in thelight path. One is placed at a crossover point on theilluminating light path. The other is placed in a pre-cisely corresponding position on the transmitted, re-flected, or fluorescent emitted light paths. For objects offinite thickness, this excludes information from out-of-focus planes, which would in a conventional microscopeconfuse the image (White et al., 1987). This deblurringof the image is the consequence of a dramatic increasein the z-axis resolution (resolution along the opticalaxis).The restricted depth of field of the instrument is

effectively producing ‘‘optical sections’’which, similar tothose produced by computed tomography (CT) andnuclear magnetic resonance (NMR) scanners andNomarski Differential Interference Contrast Micros-copy (DIC; Nomarski, 1955), are non-destructive. Withthe appropriate high Numerical Aperture lenses, theseoptical sections may be fractions of a µm in depth, thusmaking it possible even to section a microorganismsuch as the yeast Saccharomyces cerevisiae into four orfive optical slices. Once a series of confocal microscopeoptical sections is viewed through a specimen, it isimpossible to regard an image formed by a conventionalmicroscope as other than a projection which is the sumof one in-focus plane with several out-of-focus planes.One of the most widely applied microscopical meth-

ods in current use is immunofluorescence microscopy(Ockleford, 1990a). This technique suffers badly fromout-of-focus contributions to the image, because as adark-ground method its sensitivity is high and incidentlight excites fluorescence at all levels of the specimenthat are penetrated. Thus, out-of-focus haze attribut-able to planes on either side of the focal plane dramati-cally degrade the image of thick specimens.The most popular design of fluorescence microscope

for biomedical use is an epi-illuminated system. In thisdesign, illumination (often from a mercury vapoursource) passes into the intermediate tube of the micro-scope and is reflected downward by a dichroic mirror at45° to the optical axis. This light enters the objectivelens from its back focal plane and is gathered to focusonto the specimen. In this reverse-light path the micro-scope objective lens is acting as a condenser. Fluores-cent light emitted from a fluorophore in the specimen atthe point of focus of the lens is captured by the apertureof the lens and runs along a reciprocal path back intothe intermediate tube and up to the dichroic surface.Since the fluorescence is of a different wavelength(usually longer than the excitatory illumination), it istreated differently by the dichroic, which now allows itto pass up into a prism which directs the imaging beam

154 C.D. OCKLEFORD ET AL.

to the eyepieces. A key element of this design is thatcondenser and objective functions are carried out by thesame lens. Illumination and imaging light paths aretherefore necessarily perfectly aligned and it is simplerto satisfy the confocal conditions required for a CLSMmodification. In this sense, then, a widely used format—the epifluorescence microscope—was pre-adapted foradoption of the confocal principle. It was also a systemwith a great deal to gain by the adoption of the principle(Ockleford, 1995). Compare, for example, the conven-tional immunofluorescence micrograph presented asFigure 2 of Ockleford et al. (1981) with the series ofoptical sections forming Figure 3 of Bradbury andOckleford (1990). Unsurprisingly, by far the most com-mon current application of confocal technology is inepifluorescence. There are, however, several usefulexamples of reflectance applications in the literature(e.g. Sarafis, 1990), where in practice the z-axis resolu-tion can extend down as far as about 0.4 µm, which isslightly better than that achieved so far with epifluores-cence (approximately 0.7 µm); transmitted light confo-cal systems have also been constructed. The theoreti-cally calculated maximum full-width half-maximumresolution possible using confocal optics (Wilson, 1990)is 0.14 µm (lateral) 0.23 µm (depth). The advantages ofusing confocal technology to restrict the depth of aregion examined are not restricted to microscopy, otheroptical-analytical methods such as confocal laser Ra-man spectroscopy are also in development (Puppels etal., 1990).

DetectorsA variety of detector systems, including photomulti-

pliers and CCD cameras, are used to sample the imageinformation produced by the CLSM. These are capableof achieving high levels of sensitivity with extraordi-nary dynamic range compared with conventional photo-graphic detector systems. Objective sampling of weaksignals with high noise levels can be undertaken usingphotomultipliers in photon counting mode.

Digital ImagesThe intensity of the emitted fluorescent or reflected

light entering the detector (in this instance, a photomul-tiplier) leads to an electrical signal varying with timeand, hence, position within the specimen. This outputfrom the detector in its usual mode of operation is ananalogue signal which is passed to an analogue-to-digital converter and thence to a frame grabber associ-ated with a microcomputer, where a pixelated image isdisplayed and stored as a digital image. These areextensive arrays of data which might take up 0.3Mb ofmemory to store one monochromatic image which is anarray of 768 X 512 pixels recording a grey scale valuefrom 0–256 at each pixel. Digital images are secure inthe sense that they are stored more or less instanta-neously and can be quality controlled at the microscopysession while the specimen is still under observation.The digital format is extremely suitable, for it enablesthe power of image processing to be applied readily.Image processing includes three fields, all of which offeropportunities for microscopists (Niblack, 1986):Image Enhancement Only a few of the wide range

of possible enhancement techniques will be cited asexamples. These include background subtraction, en-

hancement of contrast, improvement of signal-to-noiseratio by averaging, application of filters to eliminatenoise generated in the amplification system of detec-tors, and the enhancement of particular features, suchas the edges of structures, are easily achieved.ImageAnalysis Image analysis can include automa-

tion of scaling functions to calculate magnifications orto make linear measurements typical of those made bymicroscopists for over 100 years, but it can also be usedto achieve more sophisticated measures with astonish-ing ease. Commonly, one might segment the image toisolate areas, including pixels within a particular greyscale (fluorescence intensity) range to calculate thearea as a proportion of the area of the tissue as a whole.Images can also be segmented according to other crite-ria, such as size and shape of the contained profiles.In conjunction with particle analysis programmes,

various features can be quantitated. These includecircularity, roughness, maximum and minimum Feretdiameters, orientation, centres of mass, etc. Images canbe added to and subtracted from one another andmanipulated according to a variety of mathematicaltransformation procedures. The images are also ca-pable of being imported directly into automated stereol-ogy packages which facilitate the quantitation in threedimensions of many features in irregular tissues(Howard et al., 1993). Examples of stereological mea-surements of human placenta are well represented inthe literature. These include volume fraction, harmonicmean thickness, surface density, and star volumes(Addai and Ockleford, 1994; Mayhew, 1992). Since theaccuracy of stereological methods is affected by sectionthickness and variations in section thickness, the abil-ity to easily vary the thickness of a confocal opticalsection by varying the confocal aperture diameter is aninteresting and helpful property. Confocal optical sec-tions are of more uniform thickness than mechanically-generated frozen, resin, and wax sections for lightmicroscopy. Thus, there is an indication that moreaccuracy will be obtainable from stereology of confocalmicroscope images.Rendering The rendering of digital images allows

them to be viewed in a more accessible form. Sensoryphysiology teaches us that we are ourselves creatureswhose sensory modalities employ detectors with ratherunique characteristics and limitations. We cannot dis-criminate the 256 grey-scale steps in a typical digitalimage displayed on a grey scale monitor. We are, in fact,restricted to about 50. If the image is rendered as apseudocolour display using a look-up table (LUT) thisincreases to about 200 because our colour contrastdiscrimination is so much better. The plain fact is that asimple pseudocolour rendering can uncover detail inmonochromatic imageswhich previously had lain unper-ceived.A series of confocal optical sections can be melded

digitally to produce an extended focus series. This issuperior information to the conventional image de-scribed above as a projection of one in-focus image andseveral out-of-focus images.Stacks of serial optical sections sampling a thick

specimen are known as a z-series. They are automati-cally in mutual fiducial registration, provided that thespecimen is mounted immobile on the microscope stageat the time the series is collected. In simple terms, this

155EXTRA-EMBRYONIC MEMBRANE CONFOCAL MICROSCOPY

means that the stack is neat and precise, with the partsof the specimen contained in adjacent sectionsmaintain-ing the original relationships which pertained in thewhole tissue. When digitised, these stacks of numericalinformation can then be said to describe a three-dimensional structure where the structure is sampledas a series of small volumes which are defined by thepixel dimension times the spacing between adjacentsections (Carlsson et al., 1985). This volume, oftenroughly the shape of a house brick, is known as a voxel.Calculation of projections through voxel arrays allowsinteresting rendering possibilities.The array, recalculated from a perspective at 90° to

the original optical axis, can be displayed as an x,zprojection (another form of lateral view). Calculation ofprojections from points which change by equiangularamounts and displaying these in sequence using appli-cations such as Thru-Viewy and Voxel Viewy give theimpression of a solid image being rocked or rolled oreven rotated in front of the viewer.The degree to which greater resolution can be ex-

tracted from light and electron microscopes has beendramatically extended recently with the announcementthat resolution beyond the ‘‘information limit’’ can beextracted from microscope images by using a processcalled ‘‘aperture synthesis’’ to create coherent microdif-fraction patterns as a function of probe position. Theseallow the preservation of phase information throughmaking the phase differences visible at the point ofoverlap between neighbouring pairs of diffracted beams.This approach to solving the ‘‘phase problem’’ allows thecorrection of the image for lens aberrations which arethe limiting factors which account for the fact that theconventional resolution of microscopes is so muchgreater than the wavelength of the beam they use forillumination (Nellist et al., 1995).

APPLICATIONS TO THE STUDY OFEXTRA-EMBRYONIC MEMBRANESChorionic Villus Structural Polymers

The soft-tissue components of the human placentahave an elaborate architecture which offers an ex-panded surface for exchange, secretion, and absorption(Dearden and Ockleford, 1983). This architecture, likethat of the gut, is villous but the villi are distributed ina series of arborising structures known as the villustree, rather than forming incursions into the hollowlumen of a gut tube. The tree is markedly anisometricand in large part is ultimately stabilised mechanicallyby fibrous components of the extracellular matrix coreand hydrostatic mechanisms related to fetal vascula-ture, maternal haemodynamics, and the water-holdingproperties of extracellular matrix ground substancemolecules such as the glycosaminoglycans. During itsdevelopment, the anisometry is defined by cytoplasmiccomponents and initially at least during the process ofsprouting of new villi these must be trophoblasticcomponents. We and others have used fluorescencemicroscopy to define the location of several of thesemechanically important components. This has becomepossible at higher resolution in whole villus prepara-tions through confocal microscopy, where previouslyout-of-focus blur was markedly limiting (Bradbury andOckleford, 1990; Ockleford et al., 1981). Using confocalmicroscopy and interference microscopy (Ockleford,

1990b), we were able to define cytoskeletal componentsof an apical syncytioskeletal layer and a basal syncytio-skeletal layer of cytoplasm with high refractive index,high dry mass content and a concentration of anti-keratin immunoreactivity.

Structure and Integration of AmniochorionLayers

The study of amniochorion structure is an importantfundamental activity, which will ultimately define indetail the basis of the mechanical strength of the tissueat the molecular level. We need to understand thenature of this strength so that we can approach thephysiologically important processes of parturition,which lead to amniochorion-yielding during the processof rupture. The pathology of conditions such as pre-term premature rupture of human fetal membranes,which currently is the major causal factor leading toprematurity and perinatal mortality, are also of compel-ling interest for rational approaches to minimisingthese losses.Localisation of several polypeptides which form struc-

tural polymers in the amniochorion has been achievedwith higher resolution than possible using conventionalmicroscopy. We have mapped these polypeptides usingconfocal laser scanningmicroscopy and immunofluores-cence preparations. These reveal the embryologicalorigins of cells (Ockleford et al., 1993b) and the disposi-tion of collagens (Malak et al., 1993) within the textur-ally different layers of the extracellular matrix of themultilaminous tissue. The very dense acellular layerknown as the compact layer is rich in the high-tensilestrength giving Type I and Type III collagens, whereasthis layer is devoid of detectable Type IV collagen (Fig.1a, b, c).Type IV collagen is expressed in the amniotic epithe-

lial and chorion laeve basement membranes as ex-pected, but is known to have a widespread expressionother than at basal lamina sites in placental villi(Nanaev et al., 1991). We have also shown that it isexpressed in the extracellular spaces between the tro-phoblast cells of the stratified layers of the chorionlaeve. It is also present in the extracellular matrix ofmesenchymally derived tissue layers, such as the fibro-blast and reticular layer in the form of microtrabeculae(Fig. 1c, Fig. 2b; Ockleford et al., 1993a). These novelstructures, which we have described and rendered asextended focus projections from optical sections, arerelatively large plaque-like entities which may act asnodal points in the network of mixed polymers formingthe mechanically integrated web of the extracellularmatrix. Certain glycoproteins appear to map coinci-dently with the type IV collagen distribution through-out much of the tissue (Fig. 2a). A second new confocalimmunocytochemical finding is the type IV collagen-associated series of spongy coils of the spongy layer(Fig. 2b; Ockleford et al., 1993a) which we have sug-gested may make and break contact like Velcro or twospring fasteners, so maintaining overall mechanicalcontinuity in the amnion, yet allowing the necessarylocal positional adjustments required during deforma-tion. These were initially rendered as 3-D reconstruc-tions from z-series of confocal optical sections. Confirma-tion of their existence was later gained by independent


Fig. 1. Cryosections of the similar blocks (b–e the same) of termhuman amniochorion tissue reacted in indirect immunofluorescenceprotocols with different first-step antibodies and made visible usingfluorescein isothiocyanate labelled second-step antibodies. The imageswere confocal optical sections sampled from within the frozen sectionand rendered as pseudocolour micrographs using the look-up table(LUT) known as Geog (Biorad). The LUT is revealed by the wedgeshown to the bottom right of each panel; the greatest intensities are atthe top unless stated to the contrary. a)Anti-type I collagen immunore-activity. The major site of expression is the compact layer of theamnion. The fluorescence intensity is encoded by the pseudocolour: seewedge. Bar represents 100 µm. b)Anti-type III collagen immunoreac-tivity. Major sites of expression occur throughout the tissue, with theexception of the chorion laeve epithelium (c) and the amnioticepithelium (a). Note the strong expression in the cores of degeneratevilli (d).We have interpreted these as anchoring structures similar tothe ‘‘peg and socket’’ interaction at the dermo-epidermal junction inskin. The fluorescence intensity is encoded by the pseudocolour: seewedge. Bar represents 100 µm. c) Anti-type IV collagen immunoreac-tivity. Has a classical distribution in amniotic epithelial basement

membrane and chorion laeve trophoblast basement membrane, but isalso present in the extracellular spaces between the trophoblast cells(arrowhead). The distribution in the mesenchyme is not well shown atthis magnification. The fluorescence intensity is encoded by thepseudocolour: see wedge. Bar represents 100 µm. d) Anti-type Vcollagen immunoreactivity with some similarity to type III collagenseen in Figure 1b. The fluorescence intensity is encoded by thepseudocolour: see wedge. Bar represents 100 µm. e) Anti-type VIcollagen immunoreactivity has a similar pattern of distribution to typeIII collagen seen in Figure 1b. The fluorescence intensity is encoded bythe pseudocolour: see wedge. Bar represents 100 µm. f) Anti-type VIIcollagen immunoreactivity. This is much the simplest pattern ofcollagen expression in the tissue. In this section there are three turnsof the roll, each containing amnion, chorion, and decidua, which arenot visible owing to the general lack of specific immunofluorescence.Only the three concentric turns of the amniotic epithelial basal laminaare immunoreactive with the LH7.2 anti-type VII collagen antibody(arrowheads). The fluorescence intensity is encoded by the pseudoco-lour: see wedge. Bar represents 250 µm.


means using freeze cracking and scanning electronmicroscopy (Fawthrop and Ockleford, 1994).The distribution of type V (Fig. 1d) and VI collagen

(Fig. 1e) have also been mapped.Cytoskeletons Immunocytochemical study has re-

vealed that the expression of mesenchymal and epithe-lial typical intermediate filament proteins are comple-mentary in their distribution (Figs. 2c, 3, 5b).Immunoreactivity for the muscle lineage intermediatefilament protein desmin indicates the differentiatedmotile properties and myofibroblast-like characteris-tics of most of the cells of the fibroblast and reticularlayers (Fig. 5b). These findings, together with freeze

crack data, suggest that the spongy layer is an activeshear plane in the tissue. There is, thus, now a poten-tial rational explanation for the healing of certainmembranes which are known to have ruptured because

Fig. 3. An anti-keratin immunofluorescence preparation of twoamniotic epithelial cells rendered using the same pseudocolour LUTgeog (Biorad) shown in Figure 1. The specimen has been imaged andthe scaling of the brightness has been increased so that the peri-nuclear keratin shell is imaged green. Greater immunofluorescence(red) is seen under the apical domes of these cells and this intensityincreases laterally toward the edges of the terminal web. Note,however, that at the junctional complex region the intensity dimin-ishes sharply (arrowheads). Desmosomal components occupy thesespaces. The ability to use colour contrast to define cellular compositionin this way at high resolution allows one to build up an understandingof the way mechanical integration is continuous between and throughcells. Bar represents 25 µm.

Fig. 2. Optical sections taken from frozen sections of tissue pre-pared in indirect immunofluorescence protocols. The pseudocolourmicrographs are rendered using the LUT autumn (Biorad): see wedge.The greatest intensities are to the top of the wedge. a) The basementmembrane glycoproteins laminin and nidogen are described fully inSmith and Ockleford (1994). Here, the typical distribution of nidogenis shown . It appears to be similar to that of type IV collagen.Interestingly, the highest levels of fluorescence indicating the presenceof nidogen are seen in the amniotic epithelial (a) and chorion laevetrophoblast epithelial (c) basement membranes and in marked stria-tions (arrowheads) which are observed in the decidual layer. There aretwo of these in this optical section. The fluorescence intensity isindicated by the pseudocolour: see wedge. Bar represents 50 µm. b)Athigher magnification, the immunoreactive sites in fibroblast (f),spongy (s), and reticular layers (r), the mesenchymal derivatives,revealed by anti-type IV collagen antibodies, are detected. Thisnon-classical distribution includes the spongy coils to either side of thezone of separation (arrowheads), the spongy layer, and structureswhich we have called microtrabeculae, which are expressed here assmall foci of immunofluorescence. Three-dimensional reconstructionsof microtrabeculae have been reported elsewhere (Ockleford et al.,1993a). The fluorescence intensity is indicated by the pseudocolour:see wedge. Bar represents 10 µm. c)Anti-keratin immunoreactivity isepithelial in its distribution. The basal trophoblast cells have a strongmarginated expression of the protein, shown slightly magnified in theboxed inset. The fluorescence intensity is indicated by the pseudoco-lour: see wedge. Bar represents 100 µm.


Fig. 4. The concept of super-resolution is illustrated by these twoimmunofluorescence images, which show desmosomal proteins whichanchor keratin in the membrane and make contact at the membranewith similar structures in neighbouring cells. These structures are ofthe order of 0.1 µm in diameter (below the limits of xy resolution of thelight microscope). However, the intensity of the light they emit isabove the sensitivity limit. Thus, if the individual organelles areseparated by distances greater than the point resolution, they willappear as separate punctate elements in the digital image. This effect,described as an ‘‘optical splash’’ (W.B. Amos, personal communication)can be used to gain an idea of organelle distribution, because as thecrowding of organelles increases, the punctate nature of the immuno-fluorescence becomes transformed into continuous labelling. a) The

pattern of immunoreactivity for desmoglein antibodies is restricted tothe surfaces of the cells of both of the epithelia in the tissue. Theamniotic epithelium (a) shows basal punctate and apical continuousepifluorescence, indicating reduced spacing at the junctional complexwhere the keratin immunofluorescence is locally reduced. b) Thepattern of desmoplakin immunoreactivity is very similar. Note thathere in the chorion laeve trophoblast epithelium the incidence ofpunctate profiles is higher at the basal side of the epithelium (uppersurface in this illustration). The pattern becomes more disorganizedon the decidual side (d) as the incidence drops unevenly. This and otherevidence indicates that the basal layers of this compound epitheliumare better mechanically integrated than the apical layers. Bar inframe b 5 100 µm.

of the observed loss of a small amount of amniotic fluid.Either mutual sliding of these layers or their constric-tion in purse-string fashion may be responsible forthese cases of healed rupture.The differential immunoreactivity of the cells con-

tained in the mesenchymally derived layers with re-gard to anti-vimentin and anti-desmin antibodies indi-cates that the fibroblast layer is composed of at leasttwo different types of cells. Two-channel confocal workreveals that these cells are also differentiated intosubgroups on the basis of their immune function via theexpression of different Fcg receptor isoforms (Brightand Ockleford, 1994).The identification of the desmosomal proteins desmo-

plakin and desmoglein (Figs. 4a and b) has beenachieved at the super-resolution level. Here structureswith sub-resolution dimensions emit an expandingsphere of fluorescence, which is detected by the sensi-tive photomultipliers against the dark background. Thedistribution of the fluorescence related to desmosomesand the associated intermediate filament protein kera-tin (Figs. 2c, 3, 4a and b, 5b) has highlighted theimportance of the basal two-cell layers of the chorionlaeve as being of greater structural importance thanthe apical layers. Pseudocolour display reveals a circum-nuclear concentration of keratin linking with the apicalconcentrations of the protein forming the terminal webat the apical surface of the amniotic epithelium (Fig. 3;Ockleford et al., 1993b).

Rivets and Type VII CollagenThe distribution of type VII collagen which we have

observed in this simple epithelium (Fig. 1f; Byrd et al.,1994; Ockleford et al., 1993c, 1993d) contraindicatesthe early proposals regarding its pattern of distributionin the tissues of the body, where it has been said to berestricted to compound and complex epithelia (Wetzelset al., 1991).Associated with the under-surfaces of type VII colla-

gen containing basal laminae of the amniotic epithe-lium, we have recently described ‘‘rivets’’ containingtype VII collagen which cross the compact layer andapparently thereby integrate the major layers of theamnion vertically at term (Fig. 5a; Byrd et al., 1994;Ockleford et al., 1993c, 1993d). Complete rivets havebeen optically sectioned and three-dimensionally recon-structed (Fig. 6, frames 1–18).

Fluorescent LipidAnalogue TracingAcrossChorionic Villi in Short-term Culture

The possibility that channels cross the human placen-tal syncytiotrophoblast has been raised by Kertschen-ska et al. (this volume). The morphological problemspresented by trying to define tortuous channels of smallbore using ultrathin sectioned material are notorious,especially if these are closed off, like the gut in certaincircumstances. Using whole villi, short-term culture,and confocal laser scanning microscopy on an environ-mentally controlled microscope stage we have begun tocarry out experiments using vital fluorescent tracers todefine whether such channels can be observed. Prelimi-nary observations using the fluorescent phospholipidanalogue DiI have produced interesting images whichmay support such interpretations. The hypothesis onwhich these experiments is based is that if the analogue

is allowed to diffuse laterally in the plane of the apicalmembrane passage of labelled membrane componentsto the basal surface may subsequently be observed. Ifso, this may be occurring either by transcytosis, inwhich case discrete punctate and vesicular trails shouldbe seen from apical to basal surface, or it may be thattracking along the walls of tortuous tubes will revealthe presence of channels even if these are closed andnon-functional in the sense that there is no flow throughthem at the time. Our preliminary results, obtained bymicromanipulated application of the DiI and express-ing a droplet from the tip of a micropipette so that itmade local contact with a portion of apical syncytiotro-phoblast membrane, do reveal lateral diffusion over theshort period of subsequent observation. Initially, theapical membrane is labelled over a wider range, andafter this the basal membrane, too, appears to incorpo-rate the probe. The patterns which are observed cyto-plasmically are complex and hard to classify, but atleast in some experiments we have demonstrated struc-tures which may be interpreted as apical to basalstrands or tunnels (Fig. 7, frames 1–18). These struc-tures were z-series optically sectioned and convertedinto 3-D reconstructed images using the Thru-Viewysoftware (Biorad).

DISCUSSIONHow Effective Is the Method?

Like all experimental techniques, confocal laser scan-ning microscopy has a defined area of application.However, we have found that this is enlarging and thatthe advanced microscope is frequently the instrumentof choice even in straightforward situations, such as theobservation of an immunofluorescently labelled frozensection where the only obvious advantage is the simpli-fication of the recording of images.The examples listed here include types of data obtain-

able in no other way. They show evidence of structuresobserved for the first time that would probably havebeen disregarded if investigated by conventionalmicros-copy.A final definition of the range of advanced light

microscopy is not possible, as the array of new tech-niques is still expanding.

Suggestions for FutureApplications Based onAvailable Technology; Methods Not Yet

Sufficiently Evolved but Currently in ViewThe conventional approaches to trophoblast research

have fallen mainly into four classes.

1) Studies which employ whole tissue that is fixedrapidly following birth or operative removal whichnecessarily fail to reveal the dynamics of the tissues’activities directly.

2) Studies on cultured cells which have lost theirnormal relationships within the tissue, and whilecapable of forming subjects of experiments to studydynamic changes under controlled conditions, aretherefore possibly not reflecting in-vivo physiologyaccurately (Loke, 1983).

3) Post-delivery techniques, such as placental perfu-sion and isolated perfused cotyledon (IPC) studies,which are difficult to make accurate and repeatable,


employ expensive quantities of media and fine-chemicals, such as tracers, and are a significanthealth hazard to the investigator (Ockleford andDearden, 1984).

Fig. 5. Two examples of dual channel studies. In the first, onlyone is immunofluorescence. In the second, the two detectors observethe fluorescence emission through different filter sets to discriminatethe different wavelength emissions of the different fluorophores. a)An indirect immunofluorescence preparation of a frozen sectionthrough the human amnion at term. One of the channels of themicroscope has been set up to show the Nomarski DIC image of thesection as a whole. Thus, grey scale contrast represents the rate ofchange of refractive index. One can clearly discern the individualamniotic epithelial cells and the contained nuclei. The only struc-tures immunoreactive with the anti-type VII collagen first stepantibody are the basal lamina of the amniotic epithelium and a rivetpassing down from it to cross the underlying compact layer. Theseare represented by the green image from the fluorescence channel,which has been merged with the Nomarski DIC image by a digitaladdition procedure. Bar represents 20 µm. b) A dual channelimmunofluorescence study shows the distribution of keratin anddesmin, which is clearly complementary, with the former polypeptidebeing epithelial and the latter mesenchymal. The keratin is dis-played using the red gun of the colour monitor as the upper imageplane. This was merged using COMOSy (Biorad) software with theimage of the desmin-related immunofluorescence forming the lowerimage plane using the green gun of the RGBmonitor. With appropri-ate filters and a third detector, a triple merge can be achieved usingthe blue gun. Note that in this study the desmin-containing cells aredetectable in the cores of degenerate villi. In appropriate sections,continuity with the reticular layer is seen, thus indicating that theseapparent islands of mesenchymal tissue within the chorion laevetrophoblast may be distal sections of rod-shaped projections from onelayer into the neighbouring layer. Bar represents 100 µm.

Fig. 6. The amniotic epithelial basement membrane is immunore-active with anti-type VII collagen. At intervals, structures which wehave termed rivets pass down through the underlying compact layer adistance of some 10 µm before expanding at the compact-layerfibroblast-layer interface. We believe these to be structures whichvertically integrate the multilaminar amnion tissue.A series of opticalsections (z-series) has been reconstructed using the voxel-based 3-Dreconstruction package Thru-Viewy to produce a gallery of 18 views ofone of these rivets associated with the basement membrane. Projec-tions of the structure from a series of viewpoints sucessively displacedby equiangular amounts (in this case, 20° increments) when viewed inrapid succession are interpreted by the viewer as the structurehanging in space and rotating or rolling if the displacement is in theorthogonal plane. Bars represent 40 µm.


4) In vivo noninvasive imagingmethods (Panigel, 1986)which are currently not of sufficiently high spatialand/or temporal resolution to study cell–physiologi-cal processes.

Ethical considerations rightly preclude the vast ma-jority of potential (micro-resolution) experiments onpregnant women.The use of short-term organ-cultured villi in environ-

mentally controlled stages under observation by ad-vanced light microscopes equipped with long workingdistance fluid-immersion objective lenses and vitalfluorescent ‘‘reporter dyes’’ will permit the study ofcellular dynamics in a three-dimensional system, wherethe tissue-component’s relations are preserved in astate more similar to those of the in vivo situation. Thisoption is preferable on many grounds to options 1 and 2listed above, and is the type of experiment describedabove where DiI tracer was used.The third option listed, the large-scale isolated per-

fused cotyledon systems, which are useful but difficultmethods for the study of bulk physiology, will always belimited by the variation in the condition of parts of theextensive specimen, leading to such confounding fac-tors as leakage in transepithelial transport studies(Ockleford and Dearden, 1984). They do not lend them-selves readily to direct observation of local cellularactivity as conventionally carried out, although thisdoes now become a possibility as confocal microscopesequipped with long working distance objective lenseshave been used to examine the differentiation of cells inthe brain through the meninges in chronic experi-ments. Hence, it may be possible to set up IPC’s andobserve function in villi across the thin layer of basalplate decidua through which the cannulae of the mater-nal circuit are inserted.Vascular casting has been used effectively in conjunc-

tion with scanning electronmicroscopy to study the finestructure of the fetal-derived vascular organisation ofthe cores of chorionic villi (Burton, 1987). Sectionedtissues have been used to study villous capillaries andother placental features quantitatively using stereol-ogy (Cruz-Orive and Weibel, 1990; Gundersen andJensen, 1987; Karimu and Burton, 1994). However,voxel-based three-dimensional reconstructions of z-series through vascular beds perfused with fluorescentmaterials can be made, and the possibility that thesevoxel arrays can be utilised to generate quantitativedata from the N-dimensional image analysis packageswhich are already commercially available is attractive.

SUMMARYTo review and list the significant characteristics of a

confocal laser scanning microscope:

Advantages

1) Greatly increased axial resolution.2) Increased resolution in the object plane.3) Brighter monochromatic illumination from laser

sources.4) Greater sensitivity from detectors with increased

dynamic range.5) Compact storage of the image in digital form.

Fig. 7. The syncytiotrophoblast of human term placental chorionicvilli has an apical and a basal membrane, but no conventional lateralintercellular membranes separating the nuclei and their cytoplasmicterritories. At term, this cytoplasmic layer may be 10m2 in area(Aherne and Dunnill, 1966). The fluorescent phospholipid analogueDiI has been used in its capacity as a vital dye to trace the extent of theapical surface membrane, to which it alone was initially exposed bymeans of a micromanipulator and micropipette, expressing a smallquantity until it became contiguous with the tip of a chorionic villus.After 20 min of incubation, images such as that shown in the gallery of18 views formed by rotation of a 3-D reconstruction of a z-seriesthrough part of a whole villus in short-term organ culture may berecorded. These show labelling (fluorescence) of apical and basalsurfaces and linking structures, which could be membrane-linedtunnels of restricted dimension. These images were obtained using anenvironmental stage with Peltier temperature controller and con-tained Medium 199 gassed with a mixture of 5% CO2 and air. Barrepresents 10 µm.


6) Ability to process signals and digital images conve-niently.

7) Ability to render images in useful ways (pseudoco-lour, 3-D reconstruction).

8) Variable thickness optical sections can optimise theinput for automated stereology.

Disadvantages

1) High cost.2) Increased operator skill levels required.3) Often space-consuming.4) Limited number of imaging modes.5) Relatively slow image acquisition for extensive ar-

eas at high resolution.

Extra-embryonic membrane research has alreadygained significantly from the application of advancedlight microscope techniques, and as operator skill levelsrise and instrument design is refined we can lookforward to an increasingly productive future. In ourexperience, the advantages of using this new breed ofinstruments significantly outweigh the disadvantages.

ACKNOWLEDGMENTSWe are indebted to C. d’Lacey and T. Jefferson for

their assistance. We thank Professor Taylor and theobstetricians and gynaecologists of the Leicester RoyalInfirmary for clinical coordination. We thank D. Garrodfor providing antibodies to desmosome components andJ. Smith for his preparation of the material shown inFigure 2a. We are grateful to the MRC, The WellcomeTrust, and the Royal Society for grant support. Wethank the Journal of Anatomy for permission to repro-duce Figure 3, which originally appeared in Ocklefordet al. (1993b), J. Anat. 183:483.

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