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The use of fluorescein-conjugated monoclonal antibodies, cell culture and transmission electron microscopy to detect Chlamydia psittaci and associated lesions in experimentally infected mice.

An immunofluorescence test based on a monoclonal antibody (mAb) was used to demonstrate chlamydiae in formalin-fixed and paraffin wax-embedded tissues from 10 adult mice experimentally infected by the oral route with Chlamydia psittaci isolated from the fetal membranes of an aborted ovine fetus. Samples of lung, jejunum and spleen were examined by bright-field microscopy, immunofluorescence and transmission electron microscopy, and were cultured for chlamydia in McCoy cells. These tissues were compared with those of two control mice. All infected mice had splenic hyperplasia and two had pneumonia. The lung appeared to be the target organ for C. psittaci administered by the oral route. Chlamydiae were identified in the lungs of five mice by immunofluorescence, bright-field and transmission electron microscopy. Chlamydiae were cultured from the jejunum of two mice and the spleen of one, but could not be identified at these sites by other methods. Immunofluorescence with an anti-chlamydia mAb was useful for detecting chlamydial antigen in formalin-fixed paraffin wax-embedded samples.

Animals↗

Human chromosomes: evaluation of processing techniques for scanning electron microscopy.

Methods for scanning electron microscopy (SEM) of chromosomes have been developed in the last two decades. Technical limitations in the study of human chromosomes, however, have hindered the routine use of SEM in clinical and experimental human cytogenetics. We compared different methodologies, including metal impregnation, air drying and specimen coating. SEM preparation of human chromosomes in which osmium impregnation is mediated by tannic acid, yielded more reproducible results when compared with osmium impregnation protocols previously described. The level of osmium impregnation was systematically evaluated by imaging chromosomes in the backscattering mode. Critical point drying and a light gold-palladium coating were essential for appropriate secondary electron imaging of chromosomes. With this method, and in a preliminary quantitative analysis, we show that our SEM technique is more sensitive than light microscopy for the detection of aphidicolin-induced fragile sites. This technical approach is useful for chromosomal studies requiring resolution higher than that obtained by light microscopy. Also, it allows the use of clinical and archival chromosomal samples prepared by routine cytogenetic techniques.

Adult↗

The third dimension in renal diagnosis. Scanning electron microscopy of normal and abnormal kidney.

Scanning electron microscopy, a relatively young discipline, has been used mainly for research purposes and very seldom as a diagnostic procedure. A study was made of normal and abnormal kidneys by light, transmission and scanning electron microscopes. Normal human and rat kidneys were examined under the scanning electron microscope but cellular detail was not clearly defined. Tissue was then cryofractured, critical-point dried, sputter-coated and examined again. Scanning electron microscopy then revealed greater cellular detail. Rats were injected with a solution of 0,6% uranyl nitrate in normal saline, and cryofractured sections were examined. Pathological changes were seen in the glomerulus as well as in the tubules. Scanning electron microscopy, while not replacing conventional modes of examination, can provide a rapid third dimension in the diagnosis of renal lesions.

Animals↗

Special techniques in diagnostic electron microscopy.

The power of electron microscopy as a diagnostic tool can be amplified considerably by the application of ancillary preparative and analytic methods. Subcellular chemistry and structure can be examined by various forms of microprobe analysis and by special staining methods, including cytochemical, immunocytochemical, and negative staining. Qualitative ultrastructural examination can be augmented by morphometric analysis. Correlative microscopic survey methods can be used as a means of targeting ultrastructural investigations. This article provides an overview of the use of these special techniques in the diagnosis and classification of tumors and other selected pathologic processes.

Diagnosis, Differential↗

Scanning electron microscopy of the heart after coronary occlusion.

The scanning electron microscope was used to study the ultrastructural alterations which occur following periods of myocardial ischemia of 30 minutes and 1, 2, 5, 10, and 24 hours. The alterations were compared with those seen with the transmission electron microscope. The early changes consist of swelling of organelles and clumping of nuclear chromatin. After 5 hours, disintegration of the transverse tubular system becomes apparent, and breakdown of the connections between the nucleus and the sarcoplasmic reticulum is seen coincident with distortion of the shape of the nucleus. It is suggested that the sarcoplasmic reticulum may be a part of the "endoskeleton" of the cell and may play a role in maintaining the shape and location of the nucleus. After 10 hours, extensive membrane alterations have occurred in many of the intracellular organelles. It is concluded that the changes seen with scanning electron microscopy are consistent with those seen with transmission electron microscopy.

Animals↗

Direct observation of defect structure in protein crystals by atomic force and transmission electron microscopy.

We have examined the structure of S-layers isolated from Sulfolobus acidocaldarius using atomic force microscopy (AFM) and transmission electron microscopy (TEM). From the AFM images, we were able to directly observe individual dimers of the crystal, defects in the crystal structure, and twin boundaries. We have identified two types of boundaries, one defined by a mirror plane and the other by a glide plane. This work shows that twin boundaries are highly structured regions that are directly related to the organization of units within each crystal domain. Projection maps from TEM images have shown that there are significant differences in the final average maps has allowed us to relate high magnification views obtained by AFM to the relatively high resolution information obtained by electron microscopy and image processing.

Bacterial Proteins↗

A comparison of World Health Organization (WHO) classification of lung tumors by light and electron microscopy.

Forty-nine cases of lung tumor were initially classified according to the World Health Organization (WHO) system by light microscopy. Additional study by electron microscopy using only a minimum of ultrastructural features as criteria for each type made more precise classification of some of the poorly differentiated or undifferentiated tumors possible. Most significant was the finding of evidence of a second tumor type in nine of the 35 tumors previously classified as moderately to well differentiated thus reclassifying them as mixed tumors. Sixteen of the 49 tumors were reclassified and a more precise classification was made in five others. Only two cases of undifferentiated small cell tumors were available and no unequivocal neurosecretory granules could be found in either. Correlation of the more precise typing of lung tumors by electron microscopy with the clinical data and modes of therapy will provide necessary information for determining the usefulness of the WHO classification.

Adenocarcinoma↗

Freeze-fracture electron microscopy of lipid membranes on colloidal polyelectrolyte multilayer coated supports.

Lipid membranes were assembled on polyelectrolyte (PE)-coated colloidal particles. The assembly was studied by means of confocal microscopy, flow cytometry, scanning force microscopy, and freeze-fracture electron microscopy. A homogeneous lipid coverage was established within the limits of optical resolution. Flow cytometry showed that the lipid coverage was uniform. Freeze-fracture electron microscopy revealed that the lipid was adsorbed as a bilayer, which closely followed the surface profile of the polyelectrolyte support. Additional adsorption of polyelectrolyte layers on top of the lipid bilayer introduced inhomogeneities as evident from jumps in the fracture plane. Characteristic lipid multilayers have not been seen with freeze-fracture electron microscopy.

Colloids↗

Error rates in buccal-dental microwear quantification using scanning electron microscopy.

Dental microwear, usually analyzed using scanning electron microscopy (SEM) techniques, is a good indicator of the abrasive potential of past human population diets. Scanning electron microscopy secondary electrons provide excellent images of dental enamel relief for characterizing striation density, average length, and orientation. However, methodological standardization is required for interobserver comparisons since semiautomatic counting procedures are still used for micrograph characterization. The analysis of normally distributed variables allows the characterization of small interpopulation differences. However, the interobserver error rates associated with SEM experience and the degree of expertise in measuring striations are critical to population dietary interpretation. The interobserver comparisons made here clearly indicate that the precision of SEM buccal microwear measurements depends heavily on variable definition and the researcher's expertise. Moreover, error rates are not the only concern for dental microwear research. Low error rates do not guarantee that all researchers are measuring the same magnitudes of the variables considered. The results obtained show that researchers tend to maintain high intrapopulation homogeneity and low measurement error rates, whereas significant interobserver differences appear. Such differences are due to a differential interpretation of SEM microwear features and variable definitions that require detailed and precise agreement among researchers. The substitution of semiautomatic with fully automated procedures will completely avoid interobserver error rate differences.

Animals↗

[Possibilities and limits of electron microscopy (author's transl)].

This review evaluates the electron microscopical imaging techniques used today. Reviewed are transmission method, indirect imaging of surfaces by replica and surface decoration, and direct imaging of surfaces by means of scanning, emission, and mirror electron microscopy. For each method the respective limit of resolution and the possibilities of application are described. Especially, the methods of high-resolution and of high-voltage electron microscopical techniques will be pointed out as being actual means of investigation stressing electron microscopy up to the limits given by physics and technique.

Crystallography↗

The research center for ultra-high voltage electron microscopy at Osaka University.

High-voltage electron microscopy has shown itself advantageous for the study of natural science, including biology, but especially for materials science. The most important advantage for materials science is for in situ experiments about the detailed processes of the phenomena that occur in bulk materials. The present paper is mainly concerned with several types of in situ experiments that have been carried out in the Research Center for Ultra-High Voltage Electron Microscopy, Osaka University. The following subjects have been studied: a) fundamental problems, such as the conditions necessary for in situ experiments, functional features of specimen treatment devices, and the effects of electron irradiation; b) the dislocation behavior of crystals under various conditions; c) high-temperature behavior of refractory materials, mainly ceramic composites; d) new applications of electron irradiation effects, such as amorphization of crystalline materials and electron-irradiation-induced foreign-atom implantation; e) environment-matter interaction, mainly chemical amorphization of alloys; and f) future trends of the in situ experiment, such as combinations with Auger valency electron spectroscopy and high-resolution electron microscopy.

Crystallization↗

Some contributions of electron microscopy to the study of the rickettsiae.

Electron microscopy has provided valuable insights into the study of rickettsiae as intracellular parasites from several important perspectives. This tool has allowed researchers to delineate the fine structural features of these organisms and to show that they truly resemble free-living bacteria. Furthermore, it has been shown that there are subtle, but distinct differences in the outer envelope structure of some members of the genus Rickettsia that may explain reported differences in tinctorial properties and in their sensitivity to certain antibiotics. With Coxiella burnetii, electron microscopy has helped significantly in the characterization of the pleomorphic nature of the organism including formation of terminal bodies that resemble endospores of gram-positive bacteria. Electron microsxopy has also helped to define the relationship of the rickettsiae to their host cells. For example, ultrastructural analysis can reveal whether organisms exist free within the cytoplasm or nucleus (members of the genus Rickettsia), or whether they are bound by a phagosomal or phagolysosomal membrane (Ehrlichia and Coxiella). Finally, although all rickettsiae eventually destroy their host cell, it has been shown through transmission electron microscopy that this destruction might be mediated by different mechanisms that are specific for different rickettsial species.

Animals↗

Human SP-A and a pharmacy-grade porcine lung surfactant extract can be reconstituted into tubular myelin--a comparative structural study of alveolar surfactants using cryo-transmission electron microscopy.

Cryo-transmission electron microscopy (cryo-TEM) is a rather artefact-free method, well suited to study the alveolar surfactant system. A pharmacy grade porcine lung surfactant extract (HL-10) was mixed with human SP-A and Ringer's solution (for calcium ions), and it was shown by cryo-TEM that the tubular myelin (TM) type of structure was reconstituted. These aggregates were associated to liposomal aggregates, and resulted in macroscopic phase-separation. This phase showed a weak birefringence in the polarising microscope, which is characteristic for a liquid-crystalline type of structure. TM from rabbit lung lavage was also examined, and showed the same periodic arrangement of bilayers as alveolar surface layer from freshly cut rabbit lungs deposited directly on the cryo-TEM grids. The distance between the bilayers of TM was 40-50 nm, and an electron dense material, assumed to be SP-A, was sometimes seen to occur periodically along the bilayers, oriented perpendicularly to the tubuli. The results are consistent with the surface-phase model of the alveolar lining.

Animals↗

Chemoreception in gastropod molluscs: electron microscopy of putative receptor cells.

Scanning and transmission electron microscopy of the external surface of the gastropod mollusc Pleurobranchaea californica has revealed a new exteroreceptor, characterized by dense cilia (ac.200/cell) that project from an intraepithelial soma and exhibit dilated, discoid-shaped tips. The exteroreceptor is found in high densities (up to 5000/mm2) in areas of the body determined by behavioral assay to be chemosensitive, but nowhere else, suggesting that it is a chemoreceptor.

Chemoreceptor Cells↗

["Circular bands" on the casts of the microcirculatory bed of the human heart in ontogenesis (data from scanning electron microscopy of corrosive casts)].

By means of scanning electron microscopy of corrosive casts, applied to the human cardiac hemomicrocirculatory bed vessels, beginning from 6-9-month-old fetuses up to persons of elderly and old age, peculiarities in form, dimensions and distribution have been revealed in the wall of the arterial part of the vessels of the hemomicrocirculatory bed of so called circular strips. They are inevitable structural components in the wall of the vessels mentioned and correspond to smooth myocytes, shifted at methylmetacrilate injection.

Adolescent↗

Structure-function relationships in radiation-induced cell and tissue lesions: special references to the contributions of scanning electron microscopy and hematopoietic tissue responses.

Contributions of scanning electron microscopy to the field of radiation biology are briefly reviewed and presented in terms of an overall goal to identify and characterize the structural features of radiation-induced lesions in vital cell and tissue targets. In the context of "lesion" production, the major radiation-elicited response sequences, the types and nature of measured end points, and governing temporal and radiobiological parameters are discussed and illustrated by using results derived from both in vitro cell systems and in vivo studies that measured tissue responses from various organ systems (respiratory, digestive, circulatory, and central nervous systems). Work in our laboratory on the nature of early and late hematopathologic tissue responses (aplastic anemia and myeloid leukemia) induced by protracted radiation exposure and the "bridging effect" of repair processes relative to the expression of these pathologies is highlighted.

Animals↗

Three-dimensional architecture of the Golgi complex observed by high resolution scanning electron microscopy.

In spite of many light and transmission electron microscopic studies of the Golgi complex, many important points remain unsettled. Recently, we have devised a method of observing the three-dimensional configurations of intracellular structures by scanning electron microscopy. We have used this method to study the three-dimensional structure of the Golgi complex in several tissues of the rat, paying special attention to three problems: the overall configuration; the spatial construction of each stack, and the relationship to neighbouring organelles. The interconnected stacks which form the Golgi complex are arranged in a manner characteristic of the particular cell type. In rat extraorbital lacrimal gland the stacks form an irregular network spread throughout the cell, while in anterior pituitary cells they form a hollow sphere. Continuities were seen between cisternae within a stack, and in one case a stack actually consisted of one helically wound cisterna. We also observed slender tubules connecting Golgi stacks to neighbouring rough endoplasmic reticulum. Vesicles, presumably transport vesicles, were frequently observed on the surface of the cisternae, most commonly at the cisternal rims. However the continuities among stacks and connections with cisternae and with the rough endoplasmic reticulum demonstrated here offer alternative routes for movement of substances through the cellular endomembrane system.

Animals↗