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In this study, the experimental techniques scanning electron microscopy (SEM) including energy-dispersive X-ray analysis, atomic force microscopy (AFM) and scanning small angle X-ray scattering (SAXS) have been exploited to characterize the organization of large molecules and nanocrystallites in and around the neurocentral growth plate (NGP) of a pig vertebrae L4. The techniques offer unique complementary information on the nano- to micrometer length scale and provide new insight in the changes in the matrix structure during endochondral bone formation. AFM and SEM imaging of the NGP reveal a fibrous network likely to consist of collagen type II and proteoglycans. High-resolution AFM imaging shows that the fibers have a diameter of approximately 100 nm and periodic features along the fibers with a periodicity of 50-70 nm. This is consistent with the SAXS analysis that yields a cross-sectional diameter of the fibers in the range of 90 to 112 nm and a predominant orientation in the longitudinal direction of the NGP. Furthermore, we find inhomogeneities around 7 nm in the NGP by SAXS analysis. Moving towards the bone in the direction perpendicular to the growth plate, a systematic change in apparent thickness is observed, while the large-scale structural features remain constant. In the region of bone, the apparent thickness equals the mean mineral thickness and increases from 2 nm to approximately 3.5 nm as a function distance from the NGP. The mineral particles are organized as plates in a rather compact network structure. We have demonstrated that SEM, AFM and SAXS are valuable tools for the investigation of the organization of large molecules and nanocrystallites in the NGP and adjacent trabecular bone. Our findings will be an important basis for future work into identifying the defects on nanometer length scale responsible for idiopathic scoliosis and other growth-plate-related diseases.
Although corrosion casts commonly examined by scanning electron microscopy (SEM) offer good insights into the general arrangement of the microvasculature of certain organs, no information can be obtained from such specimens on special morphological data or dynamic events. Thus, most investigators are obliged to make their interpretations of cast structures only on the basis of indirect criteria. A synopsis, which considers the most important data in that respect, is given for the blood and lymphatic microvessels. Moreover, a methodological approach is introduced, based on the application of fluorescent resin, which allows the examination of cast structures in uncorroded specimens together with the surrounding tissue by confocal laser scanning microscopy (CLSM). When two differently stained resins are injected at different sites, e.g., Mercox-fluorescent yellow into an artery and Mercox-rhodamine into the interstice, blood vessels and lymphatics can be well distinguished. In lymph nodes, the system of lymphoid spaces and that of intranodal sinuses can be separately represented with the same technique. Applications and advantages of the CLSM method, which is a useful tool for an extended interpretation of corrosion casts in SEM, are shown in cast specimens from tongue, skin, and liver in rats. In this study, both corroded and uncorroded specimens were considered. In addition, a new approach is demonstrated which combines casting technique and fluorescence microscopy with investigations carried out under experimental conditions. This allows the examination of microvascular casts together with labelled cells in liver and spleen in a state of phagocytosis.
Electron microscopy is the only currently available technique with a resolution adequate to identify and follow every axon and dendrite in dense neuropil. Reconstructions of large volumes of neural tissue, necessary to reconstruct even local neural circuits, have, however, been inhibited by the daunting task of serially sectioning and reconstructing thousands of sections. Recent technological developments have improved the quality of volume electron microscopy data and automated its acquisition. This opens up the prospect of reconstructing almost complete invertebrate and sizable fractions of vertebrate nervous systems. Such reconstructions of complete neural wiring diagrams could rekindle the tradition of relating neural function to the underlying neuroanatomical circuitry.
There is no general agreement on the relationship between results obtained by optical microscopy (PCOM) and electron scanning microscopy (SEM) or electron transmission microscopy (TEM) for airborne asbestos fibres. However, a considerable amount of data has been produced in latter years indicating that a general correlation factor, not differing greatly from unity, can be established between the two analytical techniques if counts are limited to fibres longer than 5 microns. A study was made of the relationship between SEM and PCOM when used simultaneously for determination of background concentrations of asbestos in office buildings. Twenty-four couples of samples were collected in parallel for PCOM and SEM in different locations distributed over the various floors of two buildings containing amosite sprayed on structural parts. Asbestos and total fibre counts by SEM were performed following the Verein Deutscher Ingenieure (VDI) method 3492. For total fibre counts the European PCOM method was mainly used. Discrimination of asbestos from non-asbestos fibres was performed using criteria based on birefringence and morphological characteristics of fibres. Counts included only those fibres longer than 5 microns, diameter less than 3 microns and aspect ratio greater than 3. Both total fibres and asbestos fibres were recorded separately by PCOM and SEM. the arithmetic and geometric means of the ratio between PCOM and SEM were 1.3 and 1.1 respectively for total fibres and 1.2 and 1.1 for asbestos fibres (excluding one outlying value). The regression study showed a significant linear correlation (P = 0.01) with correlation coefficients of 0.84 for asbestos fibres and 0.52 for total fibres.
A simplified and standardized technique for close correlation between light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM) is described. Perfusion and immersion fixed tissue specimens were embedded in Epon 812 and cut for conventional LM and TEM. The Epon blocks with remaining tissue were thereafter treated with epoxy solvent (ethanol-NaOH solution) for partial epoxy resin removal only (dissolving rate approx 33 microns/h). The blocks with partially blotted tissue specimens were then critically point dried and gold coated for SEM. This method, in an easy way, allows repeated observations with LM, TEM and SEM with preserved fine structure and exact correlation. Since the technique is so simple and there is no need for special equipment the method can easily be adopted in all laboratories with basic SEM standards.
Maintenance of tear film in normal conditions is dependent on mucus layer integrity and the presence and distribution of conjunctival epithelial cell microvilli. In the present work a new methodology has been developed to gain correlative information about microprojection assessment and mucus composition, from the same specimen, by Light Microscopy (LM), Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). We have characterized the glycosidic residues secreted by goblet cells in normal human conjunctiva, by means of four lectins (WGA, ConA, PNA and SBA), conjugated with FITC for LM and with colloidal gold for TEM and SEM. The cytochemical reactions were performed on histological sections of paraffin-embedded material and on semithin and ultrathin sections of both Epon embedded material directly processed for TEM and of blocks recovered from SEM and reprocessed for TEM. WGA, ConA, PNA and SBA receptors were found to be constituents of the mucus produced by goblet cells in human conjunctiva. The granules of the so-called "second mucus system" (SMS) cells were labelled mainly by WGA. A difference in the quality of glycoconjugates between goblet cells and SMS cells has been also demonstrated. Our results provide an improved method to evaluate alterations of tear film that occur in many conjunctival diseases.
Dog prostates under defined hormonal influences were investigated by light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The normal acinar cell apices exhibited well-developed, densely packed microvilli. TEM demonstrated numerous secretory granules, indicating a high secretory activity. Following castration, the acinar cells of the dog prostate showed rounded apices. Most of the microvilli disappeared and only knob-like protrusions were seen. Castrated dogs following administration of 3 alpha-androstandiol showed fewer microvilli than the controls; however, a well-developed undulating surface could be observed. Castrated dogs given 3 alpha-androstandiol and cyproterone acetate showed almost completely atrophic glandular cells. Prostates of dogs with spontaneous benign prostatic hyperplasia (BPH) were characterized by pleomorphism of cell shapes and surfaces. The different hormonal influences induced changes of the prostatic surface structures and secretory activity.
Immune electron microscopy may be used to determine the type specificity of several common human adenoviruses. The method described utilized equine antisera and was used to serotype adenoviruses within hours after typical cytopathic effect was observed in tissue culture. Adenovirus types 1, 2, 3, 5, 7a, 8, and 11 were isolated from clinical specimens and serotyped by serum neutralization and immune electron microscopy. Five of the seven types were identified consistently and identically by the two methods. Types 7a and 11 were indistinguishable by immune electron microscopy.
Scanning electron microscopy enables extensive areas of the human placental villous tree to be viewed at high resolution in a relatively quick and easy manner. It is therefore an invaluable aid to the study of normal placental structure, and may have a potential role to play in pathological diagnosis. However, with increasing experience an awareness of the possible artefacts that may be introduced during fixation and tissue preparation is gradually developing. Some of these artefacts are common to other organs and applications of scanning electron microscopy, whereas others result from the unique structure and three-dimensional configuration of the placenta. Examples of the second category are poorly documented, and yet their occurrence may also significantly influence the appearance of the tissue as seen by light or transmission electron microscopy. This paper illustrates examples of this problem, and emphasises some of the factors that must be considered when interpreting both scanning and transmission electron micrographs of the villous surface.
The intracortical pathway of cerebellar climbing fibers have been traced by means of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) to study the degree of lateral collateralization of these fibers in the granular Purkinje cell and molecular layers. Samples of teleost fish were processed for conventional and freeze-fracture SEM. Samples of hamster cerebellum were examined by means of CLSM using FM4-64 as an intracellular stain. High resolution in lens SEM of primate cerebellar cortex was carried out using chromium coating. At scanning electron and confocal laser microscopy levels, the climbing fibers appeared at the white matter and granular layer as fine fibers with a typical arborescence or crossing-over branching pattern, whereas the mossy fibers exhibited a characteristic dichotomous bifurcation. At the granular layer, the parent climbing fibers and their tendrils collaterals appeared to be surrounding granule and Golgi cells. At the interface between granule and Purkinje cell layers, the climbing fibers were observed giving off three types of collateral processes: those remaining in the granular layer, others approaching the Purkinje cell bodies, and a third type ascending directly to the molecular layer. At this layer, retrograde collaterals were seen descending to the granular layer. By field emission high-resolution SEM of primate cerebellar cortex, the climbing fiber terminal collaterals were appreciated ending by means of round synaptic knobs upon the spines of secondary and tertiary Purkinje cell dendrites.
The morphology of granulocytes collected by continuous-flow centrifugation (CFC), discontinuous-flow centrifugation (DFC), and continuous-flow filtration (CFF) was investigated in 18 healthy donors by means of light microscopy and transmission electron microscopy. Light microscopy study of semithin sections of granulocytes collected by CFC and DFC showed minimal morphologic abnormalities, compared to granulocytes procured by CFF. Ultrastructural study of granulocytes procured by CFF showed more conspicuous qualitative and quantitative abnormalities (the most prominent being "microvilli," degranulation, and bazarre chromatin) than in granulocytes obtained by the other two methods. Controls showed that the bulk of CFF-cell abnormalities was due to the "tapping" of the filters. Factors such as the mechanical compression (plasma extractor) used in DFC method, donor pretreatment with anticoagulants and steroids, hydroxyethyl starch, and duration of leukapheresis scarcely influenced granulocyte morphology.
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A total of 525 stool specimens collected during 1 year were examined for the presence of rotavirus by direct electron microscopy (EM), enzyme-linked immunosorbent assay (ELISA), and a solid-phase immune electron microscope method (SPIEM) utilizing protein A-coated grids for anchoring of specific viral antisera. Rotavirus was seen in 187 specimens; SPIEM detected 183 (97.8%), whereas direct EM and ELISA detected 161 (86%) and 166 (88.7%), respectively. No false-positive reactions were seen by ELISA. The sensitivity of the methods was evaluated by coded investigation of a dilution series of a positive sample, with a negative fecal specimen as diluent. SPIEM was approximately 30 times more sensitive than direct EM and 10 times more sensitive than ELISA. A study was done to compare the elapsed time for recognition of rotavirus by SPIEM and EM in 25 randomly selected positive specimens. All virus-positive specimens were detected within 2 min by SPIEM, whereas up to 9 min was required for direct EM. SPIEM with protein A is a highly sensitive method, useful for rapid detection of viruses in clinical specimens. Due to the direct visualization of virus particles by electron microscopy, there is no requirement for monospecific antisera for the method.
Although water soluble tracers have been used to localize intercellular permeability barriers with the transmission electron microscope, there is the possibility of translocation or loss of the tracer during processing. This study compares the localization of lanthanum tracer in keratinized oral epithelium after routine processing with lanthanum seen after using freeze drying to avoid aqueous fixation and dehydration. The electron probe was used to identify the lanthanum tracer in the tissue and to distinguish it from other electron-dense material. After incubating small biopsies of rat palate mucosa in 1% lanthanum nitrate, specimens were either routinely processed for electron microscopy or quick frozen, dehydrated, fixed in osmium vapour and infiltrated with epoxy resin. Examination in the transmission electron microscope indicated that preservation of the freeze dried tissue did not compare favourably with that of normally processed tissue, but the distribution of the electron-dense tracer in the intercellular spaces and the extent of the penetration through the epithelia was similar in the two types of preparations. Confirmation of the tracer as lanthanum was obtained by wavelength dispersive X-ray analysis with the electron probe. The results indicate that no appreciable shift in the localization of the tracer is introduced by routine aqueous fixation and dehydration for electron microscopic examination.