Histopathofogy 1982, 6, 3-24

INVITED REVIEW

Scanning electron microscopy

K.E.CARR, P.G.TONER & K.M.SALEH

Department of Anatomy, University of Glasgow, University Department
of Pathology, Glasgow Royal Injirmary and University of Basrah,
Basrah, Iraq

K.E., TONER
CARR K.M.(1982) Histopathology 6, 3-24
P.G. & SALEH
Scanning electron microscopy
The technology of scanning electron microscopy (SEM) is described in brief. Its
application to the study of cell and tissue structure is demonstrated and the
evolution of the concept of ‘topographical histology’ is discussed. Some current
literature on the applicationsof SEM is reviewed under the headings of experimental
pathology and human pathology. While SEM has become an indispensable tech-
nique for the experimental morphologist, its application to diagnostic pathology
and cytology is still at an early exploratory stage.

Keywords: scanning electron microscopy, anatomy, topographical histology,

experimental pathology, human pathology, diagnostic cytology

Introduction

Scanning electron microscopy (SEM) has come a long way from early work, which
described the surface patterns of accessible specimens, chosen more or less at random
(Carr 1971). The technique has expanded and advanced to the stage where it adds to
biomedical science in teaching (Kessel & Kardon 1979) in experimental studies
(Hodges & Hallowes 1979, 1980, Johari 1972-1981, Hayat 1974-1978) and in clinical
applications (Buss & Hollweg 1980, Carr et al. 1980, Carter 1980). As a result, a new
biological science of ‘topographical histology’ has evolved, allowing identification of
structures by their surface features and relationships in three dimensions, rather than
by their appearances in sections. Before describing the landmarks of topographical
histology, a brief description of the technology is required, in part to explain the
procedures involved and in part to emphasize the basic principles of SEM. Scanning
microscopy does not always imply surface examination of a specimen, giving
Address for correspondence: Dr K.E.Carr, Department of Anatomy, University of Glasgow,
Glasgow G I 2.
0 1982 Blackwell Scientific Publications
0309-0167/82/010~003$02.00 3
4 K.E.Carr, P.G.Toner and K.M.Saleh

topographical detail. It can equally well involve a study of its light emitting properties
or its chemical composition, since the instrumentation of SEM is unusually versatile.

Techniques

Light microscopy can be used in the transmitted mode, as in the conventional light
microscope, or in the reflective mode, as in the dissecting light microscope, where
light reflected from the surface of the specimen is used to form a magnified image.
The use of electrons instead of light allows greater resolving power and depth of field
to be applied to the problem of studying a section or a surface. The early commercial
electron microscopes of the 1950s and 1960s were transmission microscopes, using
conventional electron optics. A condenser, objective and projector lens act on an
electron beam to produce a magnified image of any specimen which has trans-
mitted part of the beam. Such a microscope, now called a conventional transmission
electron microscope (TEM) could also be used to study surface structure, by the
examination of a thin shadowed replica of the surface, through which the electron
beam was transmitted.
The concept behind the scanning electron microscope was to use a non-conven-
tional optic system to study the specimen. The specimen is illuminated by an electron
beam, focused by condenser lenses as in a standard instrument, although the
focused spot can be made smaller than is usually possible in the conventional TEM.
The non-conventional system involves the scanning of this spot across the surface
of the specimen and the synchronous scanning of a cathode ray screen on which the
image is displayed. The contrast on the cathode ray screen is dependent on the
fluctuating signal picked up from the specimen by a collector. The collector may be
below the specimen, giving a transmitted image if the specimen is thin enough.
Since the image is produced by a scanning system, this is known as scanning trans-
mission electron microscopy (STEM). Alternatively, the collector may be to the side
of the specimen, providing a signal which gives topographical detail of the specimen
surface. Such is the widely used surface scanning electron microscope (SSEM), more
often shortened loosely to scanning electron microscope (SEM). The detector may be
biased to pick up back-scattered primary electrons, or secondaries originating from
the surface of the specimen itself. Alternatively, an X-ray detector may be employed,
or even a light detector to catch fluorescence stimulated by the impact of the electrons
on the specimen surface. The nature and position of the detector are thus of prime
importance in determining the nature of the image obtained by an SEM.

SEM INSTRUMENTS

The design of the SEM therefore depends on the interaction of a primary electron
beam with the specimen and the detection of a signal which consists of any form of
energy as a result of the interaction (Figure I). Possible signals include electrons,
X-rays, and photons : each requires a suitably designed and appropriately positioned
detector. The fluctuating signal describes the situation at successive points in space
 Scanning electron microscopy 5

and time: the image of the whole specimen is produced by integration, over a variable
time interval, of all of the space points scanned. Magnification is altered by changing
the ratio of the area of the specimen scanned, which is variable, to the area of the
screen, which remains a constant. Image quality and resolution can be altered by
varyipg the accelerating voltage of the electron source, the diameter of the beam,
the size of any aperture used, the height of the specimen from the focusing Iens and
the scan speed.
The image display on the cathode ray tube can be at television speed for area
selection and focusing, or at slow speeds, such as one frame per minute for photo-
graphic recording by a camera focused on the screen. Typical exposures are of the

Figure I. Diagram to show various possible consequences E

of beam-specimen interaction in the electron microscope.
The incident electron beam, E, strikes the specimen, S. d I
\
Pathway a represents transmitted electrons not influenced
by the specimen and pathway b represents inelastically
scattered electrons. These two signals together form the
conventional TEM image. Pathway c corresponds to
elastically scattered electrons, eliminated from participa-
tion in the conventional TEM image by column apertures.
Pathway d represents high energy back-scattered primary
electrons. The back-scattered electron image (BEI) pro-
vides atomic number contrast in the SEM. Pathway e
corresponds to the secondary electron signal emitted from
the specimen surface. In the SEM,this signal carries most
of the information on surface topography. Pathway f
represents the signal of X-rays generated from the impact
of electrons with the elements of the specimen. (Repro-
duced with kind permission from Cell Structure, 1981,
3rd edn, P.G.Toner & K.E.Carr. Churchill Livingstone, b tl
Edinburgh.) a

order of seconds to 2 minutes; the image of the screen, perhaps a thousand lines, being
built up line by line on the film during this period. Although these are slow exposure
speeds, chhnging film in the camera does not involve opening the evacuated column
as in a conventional transmission microscope. As a result, scanning microscopy is
characterized by a quick throughput of routine specimens.

PREPARATION OF SAMPLES

As the image is produced from signals picked up after bombardment by primary

electrons, it is important that there should be good conductivity within the sample
and mounting apparatus, to allow excess electrons to leak to earth. For surfaces of
specimens this is achieved by sticking the specimen firmly, with conducting glue or
paint, to a metal stub which is then coated with a conducting layer of gold. Thinner
specimens can be mounted on conventional copper grids and coated with carbon;
fixation and dehydration again depend on the type of image required. Specimens for
surface examination are either freeze-dried or prepared by aldehyde fixation followed
6 K.E.Carr, P.G.Toner and K.M.Suleh

Figure 2. a Low magnification SEM of a cluster of cells from a pleural mesothelioma, showing the
rounded discrete aggregate formed by cells in an effusion. b A higher magnification shows details
of the irregular microvilli and the clefts between the surface territories of individual cells. c TEM
of the same specimen showing a similar cluster of mesothelioma cells, with bulging surfaces covered
by irregular elongated microvillous projections. Comparison of SEM with TEM images gives a
fuller picture of morphology than either technique alone.
 Scanning electron microscopy 7

by critical point drying from the last solvent. Thinner specimens may be used, such
as dewaxed sections or resin supported sections. Naturally, most investigators use
parallel samples for light, TEM and SEM to allow comparison of the results (Figure
2). A recent development is an increasing reliance on techniques which allow correla-
tive work to be done on the same specimen with SEM, light microscopy and TEM
(Carr et al. 1981c).

Topographical histology

CELLS AND TISSUES

The term ‘topographical histology’ embodies the concept of histological images which
convey the three-dimensional relationships of cells and tissues. The average surface
scanning electron micrograph gives a large amount of topographical information.
A summation of results can be achieved by producing stereopairs, by taking two
pictures at different specimen tilt angles. Alternatively, information on the usual
dispositions of the ceNs in an epithelial sheet can be built up from many views of
similar specimens. In conventional histology, three dimensional information is often
obtained by building up a series of sections which run parallel to the plane of examina-
tion. In topographical histology, three-dimensional information is already available
on the image and added information is obtained by tilting and rotating the specimen
and imaging it from different angles.
The recording of all of the information is restricted only by the inability of a two-
dimensional page to convey information on spatial relationships. In conventional
histology, the information can sometimes be summarized usefully in a diagram. As
topographical histology becomes better understood, it may be that maximum informa-
tion transfer may also be achieved by using diagrams (Figure 3). No matter how the
information is stored and transferred, SEM provides a wealth of new information
on the topographical histology of cells and tissues, running parallel to conventional
histology.
In epithelial cells, which are lining and covering cells, widely variable apicaI sur-
face specializations can be made out, and their distribution over a mucosal surface
can be displayed (Figure 4). Squamous cells have surface microridges (Figure 4b),
while cuboidal or columnar cells almost always have a few surface microvilli, sug-
gesting an absorptive role. Specialized cuboidal or columnar cells may be covered
with a mat of microvilli, as in small bowel, (Figure 4c) or a carpet of cilia, as in trachea.
Serosal cells have longer, sparse microvilli and occasional cilia. Epithelial cells in
glands can often be identified by the presence of masses of granules, causing the cell
to bulge. Ducts can be identified as tubes, lined with regular epithelial cells firmly
adhering to each other.
The topographical differences between tightly packed epithelial tissue and the
more dispersed connective tissues emphasize their different functions. Unspecialized
connective tissue can be seen to contain identifiable cells, such as fibroblasts, along-
side bundles of fibres and fibrils. Blood vessels (Figure 4d) and nerve bundles can be
8 K.E.Carr, P.G.Toner and K.M.Saleh

Figure 3. Diagram of simple columnar epithelium, drawn from several SEMs of small intestinal
mucosa. The apical surface is covered with microvilli, the lateral surface with flat interdigitating
processes and the basal aspect shows the circular shape of the cell bases, with wisps of connective
tissue connecting them and capillaries lying beneath.

identified in the connective tissue. Specialized connective tissues such as cartilage

and bone (Figure 5a) have distinctive topographical features, with alternating cellular
and solid components. Topographical information on blood and lymph cells is
obtained by isolating the cellular elements. Red blood cells have the familiar bicon-
cave disc shape and have a smooth cell membrane. Lymphocytes are round and their
cell surface varies from fairly smooth to microvillous. Monocytes, like macrophages
elsewhere (Figure gb), vary in shape depending on their state of stimulation. Their
cell membrane is often characterized by the presence of ruffles. Although less is
known so far about the surface features of muscle and nerve, the regular periodicity
of the underlying myofibrils can be seen in striated muscle cells. Nerve bundles can
be recognized and the close packed nature of spinal cord and brain tissue can be
recorded.

SYSTEMS
Since the systems of the body are made up of Cells and extracellular material, all with
a distinctive topography, three-dimensional information can be collated by SEM
to summarize the structure and function of these systems. The conducting and gaseous
exchange parts of the respiratory system can be represented by the contrasting
topographical images of ciliated epithelium (Figure 5c) and red blood cell profiles
 Scanning electron microscopy 9

Figure 4. a SEM of human endometrium, showing the distribution of ciliated cells, by comparison
with secretory cells, covered with microvilli. b SEM of squamous cells, covered with microridges,
from human cervix. c SEM of microvillous border of small intestinal absorptive cells. d SEM of
capillary plexus of small intestinal villus. The connective tissue has been exposed by ultrasonic
disintegration.
10 K.E.Carr, P.G.Toner and K.M.Saleh

Figure 5. a SEM of cut surface of human bone. A Haversian canal and several lacunae can be seen.
b SEM of human macrophage from serous effusion, showing the prominent surface ruffles which
characterize this cell type. c SEM of bronchus showing the mucociliary specializations appropriate
to the conducting part of the respiratory system. The blebs are the prominent apices of secretory
cells, lying in between tufts of cilia. d SEM of alveolar wall showing a surface view of the blood-air
barrier. The extreme thinness of the alveolar wall is indicated by the visible profiles of red blood
cells, bulging through the wall.
 Scanning electron microscopy II

(Figure 5d) seen through the thin alveolar wall (Andrews 1979a). In the gastro-
intestinal system, functions of protection, secretion and absorption in oesophagus,
stomach and small bowel can be summarized by images of microridge-covered
squames, pit mouths and villi respectively (Toner and Carr, 1979). Features of the
reproductive system include the specializations of sperm for movement and the
Fallopian tube for cilium-assisted transport (Ferenczy 1980). Complicated surface
structures in the urinary system are linked with filtration in the glomerulus (Andrews
1979b) and specialized protection in the bladder (Hodges 1979).
In all these normal systems, it is becoming increasingly recognized that the func-
tion of cells and tissues is displayed on the common walls they share with their
neighbours as surely as in their interior furniture.

P A T H O L O G I C A L LESIONS A N D RESPONSES

Just as the basic cells, tissues and organs now have identifiable topographical patterns,
so in pathological situations the state of the health or disease can be inferred from
surface patterns. The presence of unusual types or numbers of flora or parasites can
be quickly detected. Even surface debris can be informative. The normal clean or
mucus-covered surface can give way, as in ulcerative colitis, to a surface covered with
exudate, with recognizable components of fibrin and blood cells (Figure 6a). At
tissue levels, ulcer craters in an epithelial surface are occupied at an early stage by
connective tissue full of cells such as macrophages and lymphocytes, indicating the
presence of inflammation, and latterly by an irregular epithelial covering (Figure 6b).
Abnormal or irregular epithelium is seen at the edge of the ulcer, while regions
distant from the ulcer are comparatively normal (Carr et al. 1979). Marked disorgan-
ization of mucosal tissue can be seen in tumour growth (Figure 6c) and other abnormal
situations (Figure 6d). Their surface patterns are quite dissimilar from the regular
surfaces of normal mucosa (Carr & Toner 1972, Anderson & Withers 1973, Friberg
1980, Carr 1981).
While the situations described above, illustrating disease at tissue level, have
topographical manifestations easily seen with the SEM, the technique can also illus-
trate the responses of individual damaged cells which are responding to trauma. Cell
surfaces may show extra ruffles, fewer microvilli or damaged cilia. Abnormal cells,
such as giant cells seen in some radiation-damaged mucosae, were first identified by
SEM by their grotesquely abnormal shapes (Carr et al. 1981a).

Applications of SEM technology

E X P E R I M E N T A L PATHOLOGY

The experimentalist can exploit the various imaging modes of the SEM and can
provide the stimulus for new technological developments. For example, the ultra-
high resolution which can be attained by the use of the field emission gun has been
used for the study of intracellular organelies, following modified preparation tech-
Figure 6.a SEM of human colonic mucosal surface, from a patient suffering from ulcerative colitis.
Cells of the surface exudate, strands of fibrin and bacteria are seen lying on the partly obscured
epithelial surface. b SEM of rat small intestinal mucosa, showing the edge of a recently re-epithelial-
ized ulcer crater. An abnormal villous pattern is seen at the top left, contrasting with the irregular,
pitted epithelial surface of the newly-covered ulcer base. c SEM of human colonic biopsy showing
part of a papillary adenoma, characterized by irregular clefts and folds. d SEM of human colonic
surface from a patient suffering from Hirschsprung’s disease. An abnormality of the surface is
indicated by a criss-crossing network of surface ridges, indicating perhaps an abnormal cell extrusion
process.
 Scanning electron microscopy I3

niques (Tanaka 198I). The resulting display of three-dimensional subcellular detail

could previously only be grasped through artists’ reconstructions, extrapolated from
high resolution TEM studies of thin sections. Two other rapidly developing areas of
SEM technology are back-scattered electron imaging (BEI), and X-ray microanalysis,
both of which can help to introduce a new functional dimension into the SEM
image. BE1 introduces atomic number contrast, while X-ray analysis allows the
identification of the spectra of individual elements within the specimen. If concentra-
tions permit, elemental distribution maps can be superimposed on the corresponding
secondary or back-scattered electron image (Carter 1980, Abraham I 980). Detailed
applications in experimental pathology include studies of kidney (Bulger 1980)
and muscle function (Somlyo, Somlyo & Shuman 1979), and of cellular injury
(Trump & Berezesky 1979). In a recent study of experimental myocardial infarc-
tion, Osornio-Vargas, Berezesky & Trump (1981), demonstrated the early inflow of
Na+ into the damaged myocardium. The subsequent rise in the intracellular Ca2+
concentration appeared to correlate with irreversibility of the injury.
The majority of experimental studies, however, still concentrate on the use of the
secondary electron image. In general, morphologists are now well aware of the need
for parallel light microscopy, TEM and SEM observations, in recognition of the
limitations of each of these methods used on its own. The published applications of
scanning microscopy are now too numerous for any single review, but in the follow-
ing section a few examples have been selected to indicate the diversity of uses of this
technique.

TUMOURS

Ogawa, Medline & Farber (1979) have described an in-vivo model of hepatic carcino-
genesis in the rat. The well-synchronized early steps of malignant change are accom-
panied by various alterations of the cell surfaces, most of which, however, are best
demonstrated by conventional TEM. Heckman & Olson (1979) described surface
changes in an in-vivo system of oncogenesisin a population of epithelial cells originat-
ing from the respiratory tract. These changes were related to disturbances of the
cytoskeleton or of cellular adhesion mechanisms. Kramer & Nicolson (1979) used
both time-lapse cinephotomicrography and SEM in the investigation of tumour cell
interactions with monolayer cultures of vascular endothelial cells. A corresponding
in-vivo study by DeBruyn & Cho (1979) demonstrated a specific interaction between
the cells of subcutaneous deposits of a myelogenous tumour and the endothelium of
blood vessels. The tumour cells were shown to enter the circulation by forming
temporary migration pores, through which three or four cells at a time would cross
the vessel wall. Finally, Mennel(1980) has used SEM to help in defining the cellular
derivation of various experimental tumours of the rat nervous system, maintained in
a short-term culture.

VESSELS

The lining of the vascular system represents a natural body surface which has often
attracted scanning microscopists (Buss & Hollweg 1977). Woolf (1979) has used
14 K.E.Carr, P.G.Toner and K.M.Saleh

scanning images to illustrate endothelial lesions and platelet interactions in experi-

mental vascular injury. McGowan & GlIett (1980)~studied the early lesions of
experimental streptococcal endocarditis in rabbits. Bacterial cells were noted in
significant numbers on endocardia1 surfaces within 10-1 5 min after inoculation, but
then became less apparent until 20 hours, when colony growth within vegetations
had progressed sufficiently to allow the organisms to reach the surface again.
In hypertension, both spontaneous (Hazama, Ozaki & Amano 1979) and induced
(Haudenschild, Prescott & Chobanian 1980, Povlishock et al. 1980)~various vascular
endothelial changes can be studied in detail by SEM. The endothelium of spontane-
ously hypertensive rats was characterized by enlargement and bulging of the endo-
thelial cells and by increased numbers of microvilli and surface pits. In cats, acute
endothelial surface changes were correlated with abnormal vascular leakiness within
minutes of the onset of acute systemic hypertension (Povlishock et al. 1980).
There is current interest in the interaction of tissues and prosthetic vascular
material, particularly in the field of microvascular grafts (Medhorn et a/. 1979,
Loisance et al. 1979, Lidman, Faibisoff & Daniel 1980). The bio-degradable, bio-
logical prostheses of Loisance et al. (1979) became endothelialized with a result
similar to normal, but without the longitudinal ridging typicai of the natural vessel.
The results with these prostheses compared favourably with those obtained by the
use of artificial materials in the same size range.

ALIMENTARY TRACT

An extensive literature on this topic has recently been reviewed (Toner & Carr 1979).
Not surprisingly, the small intestine accounts for much of the literature, but there is
now growing interest in the morphology of the large bowel and of other mucosal
surfaces.
Martin (1980) examined the gut of Nippostrongylus brasiliensis-infected rats
maintained on a low protein diet, and observed large areas of villous atrophy. Other
surface abnormalities pointed to cellular injury and leakage of blood constituents
into the gut. Hutchison et al. (1980) studied the mucosal response of Toxoplasma
gondii-infected cats, demonstrating partial villous atrophy and enlarged parasitized
enterocytes. Carr et al. (198rb) have highlighted the differences between the experi-
mentalist and the diagnostician in their approach to studies of the gut, with particular
reference to the effects of radiation injury.

RESPIRATORY SURFACES

These are well suited to SEM study, particularly in investigations of the damaging
effects of inhaled toxic substances. The effects of high oxygen concentrations, as
shown by Obara, Sekimoto & Iwai (1979), included denudation of bronchial cilia,
bleb formation at cell surfaces and desquamation after 96 hours, with flattening of the
luminal surface in the bronchioles. The short-term effects of ozone exposure in rhesus
monkeys include an acute respiratory bronchiolitis (Castleman et al. 1980)~the type
I bronchiolar epithelial cells showing the greatest sensitivity to injury. The develop-
 Scanning electron microscopy I5

ment of metaplasia and dysplasia in high dosage long-term exposure to sulphur

dioxide was followed by Morgenroth (1980) using TEM and SEM. Pulmonary
parenchymal changes have also been studied, as in papain-induced experimental
emphysema (Parra, Gaddy & Takaro 1980), in which macrophage destruction was
postulated as a pathogenetic factor.

RENAL GLOMERULUS

Jones (1977) has shown how the SEM and TEM images of normal and abnormal
glomeruli can be correlated, and has demonstrated various typical reaction patterns,
such as the increase in podocyte microvilli in hypertension. Like most specialized
cells, however, the podocyte has only a limited repertoire of response to injury, which
leads to a lack of specificity in the SEM image. Takizawa, Shigematsu & Kondo
(1979) have studied vascular casts of renal glomeruli in chronic Masugi nephritis in
rats, showing the progressive loss of communicating branches between glomerular
loops. SEM was found to be particularly useful in the detection of limited lesions;
glomeruli apparently normal on first examination revealed their involvement on care-
ful study from different angles.
Outgrowing cells in tissue cultures of isolated glomeruli were studied by Morita
et a]. (1980), who recognized four cell lines, derived, respectively, from visceral epi-
thelium, mesangium, monocytes and parietal epithelium. A distinctive modification
of Bowman’s capsule was produced by Ojeda, Garcia-Porrero & Hurle (1979) by
post-natal injections of methyl prednisolone acetate; this consisted of a cystic
abnormality of Bowman’s capsule, with apparent metaplasia of the parietal epithelium
to a podocyte pattern, with striking surface ultrastructural characteristics.

Human pathology

The title of this section has been carefully chosen to avoid the often misused term
‘diagnostic SEM’. Of the many reported studies of human disease by SEM, there
are still few which can honestly be said to offer practical assistance to the diagnostic
pathologist (Carter 1980, Carr et al. 1980, Buss & Hollweg 1980). There are, of
course, exceptions. In forensic medicine and science, for example, SEM has made a
substantial impact (Somogyi & Sotonyi 1977, Taylor & Noguchi 1979). Another
important exception is the elemental analysis of human tissues by X-ray spectroscopy.
For example, the combination of secondary electron imaging, BE1 and microanalysis
can provide conclusive identification of particulate substances causing lung disease
(Funahashi et al. 1977, Abraham 1979, Roub et a/. 1979, Sherwin, Barman &
Abraham 1979).
Bjelle, Crocker & Willoughby (198 I) described a case of pyrophosphate crystal
arthropathy in which the crystals were less than I pm in length, too small to be
recognized on polarizing microscopy of synovial fluid. The authors suggest that
crystals of this size may cause disproportionate acute symptoms and could easily be
overlooked, leading to a failure to reach a correct diagnosis.
16 K.E.Carr, P.G.Toner and K.M.Saleh
 Scanning electron microscopy I7

X-ray microanalysis of human myocardium is unlikely to become a routine

procedure, but Abraham, Higgins & Newel1 (1980) have shown that iodinated
contrast material taken up by ischaemic myocardium can be demonstrated by SEM
and X-ray microanalysis of frozen sections. Techniques such as this, and the work of
Osornio-Vargas et al. (1981), may help in the future to locate and determine the extent
of recent myocardial infarcts at autopsy.

DIAGNOSTIC CYTOLOGY

While SEM has obvious possibilities in this field (Koss & Domagala 1980, Berliner,
Janssen & McLatchie 1978), it is still of only limited diagnostic value. Gondos, Lai
& King (1979), for example, claim to recognize criteria of microvillous morphology
which help to distinguish atypical mesothelial cells from malignant cells in effusions.
In general, however, the accepted cytodiagnostic light microscopy criteria of malig-
nancy show no signs of yielding to new criteria derived from surface morphology
(Figures 7a-f).
There has been substantial interest in the SEM appearances of the neoplastic
urinary bladder (Newman & Hicks 1977, 1981) and of the uterine cervix (Rubio &
Einhorn 1977, Davina el al. 1981). Although some promising results have been
reported, there is, as yet, no agreed basis for the routine use of SEM in diagnosis.
In particular, Price, Morley & Hall (1980) have cast doubt on the often quoted
specificity of pleomorphic microvilli as markers of bladder malignancy.
In this general context, Davina et al. (1981) and Kenemans et al, (1981) have
pointed out an obvious but hitherto largely ignored pitfall of interpretation. Having
shown that the superficial and the deep aspects of flattened cells on mucosal surfaces
are morphologically different on SEM, these authors pointed out that half of all such
cells exfoliated from the surface will come to lie ‘upside down’ when examined by
SEM. This has obvious implications for studies comparing exfoliated cells with the
corresponding natural tissue surfaces, whether normal or neoplastic.

VESSELS

Bylock et al. (1979) were able to distinguish the uterine arterial endothelium of non-
smokers from that of smokers. Sheffield &’ Weller (1980), recorded the occurrence
of inelastic intimal pads in the vessel walls proximal to the necks of berry aneurysms.
With increasing age, similar, but smaller pads can be seen in many normal patients

Figure 7. a & b Low and high magnification SEM of a group of histiocytes and lymphocytes from a
human pleural effusion. The large rumed cells are histiocytes, the smaller cells with smoother con-
tours are lymphocytes. In the low magnificationview, the surrounding red corpuscles can be clearly
seen. c & d Low and high magnification SEM of mesothelial cells from a human pleural effusion,
The cell surfaces are characterized by microvilli and blebs. Mesothelial cells can show a spectrum
of surface appearances, ranging from this pattern to an almost flat and featureless surface. e & f
Low and high magnification SEM of a group of tumour cells from a malignant pleural effusion.
The primary tumour was a well-differentiated bronchial adenocarcinoma.
L
I8 K.E.Carr, P.G.Toner and K.M.Saleh

at points around and distal to bifurcations. The authors suggest that the presence of
abnormally large pads might alter the haemodynamic stresses at bifurcatiens,
predisposing to aneurysm formation at the gaps in the vascular media which are not
uncommon at these points. McCollum & Campbell (1979) used $EM to confirm the
identity of intravascular platelet aggregates, as measured by a screen atration pres-
sure technique. This appeared to provide a means of predicting the occurrence of
late postoperative hypoxaemia, presumably due to pulmonary microembolization.
The red blood corpuscles themselves have been the target of various SEM investi-
gations and their morphology in various diseases is now well documented (Bessis
I 973). Even when not immediately apparent, variations from the normal biconcave
disc shape can be more easily induced by experimental manipulation of blood samples
from patients in certain clinical conditions than in controls (Dardano & Kimzey
1979). Kucukcelebi, Barmatoski & Barnhart (1980) have demonstrated that sickle
cells adhere to vascular endothelium 10 times more readily than normal cells, and
attribute some of the microvascular pathology of sickle cell disease to this propensity.

ALIMENTARY TRACT

SEM has become a standard tool in the investigation of mucosal disease of the gut,
from the oral cavity (Reichart & Althoff, 1979) to the rectum (Kaye et al. 1979).
Initial results from studies such as these have so far failed to indicate any convincing
diagnostic advantage of SEM over conventional histopathology, although Dvorak,
Connell & Dickersin (1979) and Myllarniemi & Nickels (1980) support its use in
Crohn’s disease, suggesting that it may be of value in detecting minor abnormalities
in resection margins which are macroscopically and histologically unremarkable.

RESPIRATORY SURFACES

The uncommon conditions of familial and primary atrophic rhinitis have been
compared by Barton et al. (1980) and by Gray et al. (1980). In a family with genetic-
ally determined atrophic rhinitis, eight of 15 children were affected. In the cases
studied by SEM, loss of cilia and abnormalities in the overlying mucus blanket were
the most striking abnormalities. The onset of the condition is usually around puberty;
one 13-year-old who was clinically normal was found to have abnormal findings by
SEM. Eight months later, however, clinical atrophic rhinitis developed, suggesting
a possible predictive value for the SEM image.
Dionne & Wang (1977) studied a group of tumours of the lungs and pleura,
outlining criteria for the recognition and possible differential diagnosis of the major
types of neoplasm by SEM. Takenaga et al. (1977,1980) developed techniques for the
study of the same cell by light and SEM, and applied these to lung tumours. Meinl
& Brunner (1980) concentrated on alterations of the bronchial basement membrane
in metaplasia and dysplasia. This layer appears to display projecting connective
tissue papillae, which project into the bronchial epithelial lining and which respond
to alterations in the architecture of the mucosa.
 Scan,ningelectron microscopy 19

RENAL GLOMERULUS

The pathology of the renal glomerulus has been studied in considerable detail in a
series of 150 cases by Jones (1979), using SEM in conjunction with light microscopy,
and TEM. Although the value of the three-dimensional image is not disputed and
continued study of this problem is considered valid, Jones believes that the greatest
benefits to come so far from SEM lie in the conceptual and aesthetic supplementation
of data gained from light microscopy, immunofluorescence and TEM. Langlinais
Myers & Merrill (1980), studied 16 patients with acute renal failure secondary to
burn injuries. They were unable to discriminate, on the basis of SEM, between
cases with a favourable outcome and those with an unfavourable clinical course.

BREAST

Interest in the SEM appearances of breast ducts was stimulated by the report of
Spring-Mills & Elias (1976) that histologically normal ducts from cancerous breasts
showed distinctive abnormalities. Halter, Holt & Page (1981) have attempted to
document more fully some of the complex patterns of neoplasia, dysplasia and
hyperplasia in the human breast. Such studies are complicated by the lack of uni-
formity of histological nomenclature, by the multicentricity of breast pathology and
by the importance of physiological variations which have not yet been documented,
but which may possibly influence the interpretation of the results.

Conclusions

This brief outline has covered only a fraction of the relevant references in just a few
areas of application of SEM in the biomedical field. A few generalizations, however,
can be made.
As with many new techniques, SEM has enjoyed a ‘honeymoon’ period of wide-
spread popularity amongst anatomists and experimental pathologists. To some
extent, it is fair to say that many of the published results rely more on the visually
arresting qualities of the scanning image than on any unique information which it
secures. This, however, is only to be expected, and does not imply criticism. The
secondary electron SEM image can display, in a readily appreciated form a range
of information that would otherwise call for several light and transmission electron
micrographs, along with paragraphs of commentary. In some respects, the impact of
SEM on scientific investigation can be compared with that of photomicrography.
Both have become almost universally available to workers in the biomedical field.
Both techniques are relatively simple in their basic form, while being capable of great
sophistication in the hands of an expert. Both are fundamental techniques of illus-
tration, of use even in pedestrian studies, but capable also of revealing fundamental
scientific insights in a most effective way.
Scanning microscopy is now an integral part of the resources of the teacher of
anatomy and histology. It is invaluable in many areas of experimental pathology,
20 K.E.Carr, P.G.Toner and K.M .Saleh

making possible some investigations which would be impracticable if one were forced
to rely on conventional transmission electron microscopy. In human pathology,
scanning microscopy adds to our insight into certain conditions and offers limited
diagnostic assistance, particularly where microanalysis can be deployed. The future
role of SEM will require to be established by the continued re-evaluation of its appar-
ent failures and of its apparent successes.

Acknowledgements

The authors are grateful to their technical colleagues for assistance in this work, and
in particular to Mrs C.Watt, Miss A.Hughes, Miss C.A.Morris and Mr J.D.Anderson,
FIMLS. The use of research facilities in the Departments of Anatomy and Pathology
is also gratefully acknowledged. The work of Dr K.M.Saleh was supported by a
grant from the University of Basrah, Iraq. The scanning electron microscope used
in this work was purchased by the MRC on a special departmental apparatus grant.
The assistance of Dr H.E.Hughes, Cytology Department, Glasgow Royal Infirmary
is gratefully acknowledged.

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