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PubMed1984

High-voltage electron microscopy of capillary endothelial vesicles.

Conventional electron microscopy of thin sections through capillary walls is inadequate to discern the relationships between endothelial vesicles and their association with the cell surface. High-voltage electron microscopy of thick sections through diaphragm muscle capillaries has been employed to visualize the three-dimensional structure of the vesicular system. Stereopairs of thick sections provide for direct three-dimensional observations of samples several vesicle diameters deep. A variety of simple and compound vesicular forms are present but not all are conjoined or maintain connections with the endothelial cell surface. This contributes to the concept of a dynamic interaction between free and attached vesicular structures where fission and fusion events compartmentalize and reconnect repeatedly. Such interactions would provide for a discontinuous pathway across the capillary wall but with a higher degree of complexity than a simple shuttling mechanism.

Animals↗
PubMed1990

[Predictive value of electron microscopy studies in virological diagnosis].

The negative staining techniques in transmission electron microscopy have been used as a routine tool in viral diagnosis since 30 years. In a short review, different preparation techniques, fields of application, advantages and disadvantages of electron microscopy in rapid diagnostic are discussed. The application of electron microscopy depends on availability of other more effective methods. Some viruses, specially in faecal samples, are identifiable only by electron microscopy till now. Sensitivity and specificity are improved by immune electron microscopy comprising suspension, immunosorbent and immunogold techniques. Using rapid embedding techniques, ultrahistology is a valuable tool in identifying viruses in biopsy samples, too.

Histological Techniques↗
PubMed2005

Breaking the spherical and chromatic aberration barrier in transmission electron microscopy.

Since the invention of transmission electron microscopy (TEM) in 1932 (Z. Physik 78 (1932) 318) engineering improvements have advanced system resolutions to levels that are now limited only by the two fundamental aberrations of electron lenses; spherical and chromatic aberration (Z. Phys. 101 (1936) 593). Since both aberrations scale with the dimensions of the lens, research resolution requirements are pushing the designs to lenses with only a few mm space in the pole-piece gap for the specimen. This is in conflict with the demand for more and more space at the specimen, necessary in order to enable novel techniques in TEM, such as He-cooled cryo electron microscopy, 3D-reconstruction through tomography (Science 302 (2003) 1396) TEM in gaseous environments, or in situ experiments (Nature 427 (2004) 426). All these techniques will only be able to achieve Angstrom resolution when the aberration barriers have been overcome. The spherical aberration barrier has recently been broken by introducing spherical aberration correctors (Nature 392 (1998) 392, 418 (2002) 617), but the correction of the remaining chromatic aberrations have proved to be too difficult for the present state of technology (Optik 57 (1980) 73). Here we present an alternative and successful method to eliminate the chromatic blur, which consists of monochromating the TEM beam (Inst. Phys. Conf. Ser. 161 (1999) 191). We show directly interpretable resolutions well below 1A for the first time, which is significantly better than any TEM operating at 200 KV has reached before.

Lenses↗
PubMed2005

Visualization of the nuclear lamina in mouse anterior pituitary cells and immunocytochemical detection of lamin A/C by quick-freeze freeze-substitution electron microscopy.

We examined the nuclear lamina in the quickly frozen anterior pituitary cells by electron microscopic techniques combined with freeze substitution, deep etching, and immunocytochemistry and compared it with that in the chemically fixed cells. By quick-freeze freeze-substitution electron microscopy, an electron-lucent layer, as thick as 20 nm, was revealed just inside the inner nuclear membrane, whereas in the conventionally glutaraldehyde-fixed cells the layer was not seen. By quick-freeze deep-etch electron microscopy, we could not distinguish definitively the layer corresponding to the nuclear lamina in either fresh unfixed or glutaraldehyde-fixed cells. Immunofluorescence microscopy showed that lamin A/C in the nucleus was detected in the acetone-fixed cells and briefly in paraformaldehyde-fixed cells but not in the cells with prolonged paraformaldehyde fixation. Nuclear localization of lamin A/C was revealed by immunogold electron microscopy also in the quickly frozen and freeze-substituted cells, but not in the paraformaldehyde-fixed cells. Lamin A/C was localized mainly in the peripheral nucleoplasm within 60 nm from the inner nuclear membrane, which corresponded to the nuclear lamina. These results suggest that the nuclear lamina can be preserved both ultrastructurally and immunocytochemically by quick-freezing fixation, rather than by conventional chemical fixation.

Animals↗
PubMed2000

Sequence-specific liquid crystallinity of collagen model peptides. I. Transmission electron microscopy studies of interfacial collagen gels.

The conformation, crystal structure and self-assembly behavior of three peptides with collagen-like repetitive sequences [(1) peptide GAPGPP: (Glu)(5)(Gly-Ala-Pro-Gly-Pro-Pro)(6)(Glu)(5); (2) peptide GVPGPP: (Glu)(5)(Gly-Val-Pro-Gly-Pro-Pro)(6)(Glu)(5); and (3) peptide GAPGPA: (Glu)(5)(Gly-Ala-Pro-Gly-Pro-Ala)(6)(Glu)(5)] were compared. The peptides were characterized using transmission electron microscopy, electron diffraction, environmental scanning electron microscopy, and Fourier transform ir spectroscopy in order to determine how the molecular geometry dictated by each sequence affects the spontaneous generation of long-range ordered structures. Samples of each peptide, at ambient temperature and at 5 degrees C, were examined as films dried from aqueous solution, air-water interfacial films, and chloroform-water interfacial films. Peptide GAPGPP prepared at 5 degrees C and dried from bulk solution was found to have a collagen-like triple-helical structure. A sinusoidally textured gel, suggestive of cholesteric behavior was observed for peptides GAPGPP and GVPGPP at the aqueous chloroform interface at 5 degrees C. Peptide GAPGPA also formed a gel, but less reproducibly and the sinusoidal texture was not as well defined. The periodicities of the sinusoidal textures were reproducibly 10 microm for peptide GAPGPP, 7 microm for peptide GVPGPP, and 6 microm for peptide GAPGPA. The differences in the periodicity of the banded structure and in the crystallization behavior of the three peptides is attributed to differences in the symmetry of the preferred packing arrangement for each peptide, as evidenced by electron diffraction from crystallites that coexist with the sinusoidal gel. These differences are believed to be a measure of the effective symmetry and shape of the molecular cross section.

Amino Acid Sequence↗
PubMed1985

Mass and molecular composition of vesicular stomatitis virus: a scanning transmission electron microscopy analysis.

Dark-field scanning transmission electron microscopy was used to perform mass analyses of purified vesicular stomatitis virions, pronase-treated virions, and nucleocapsids, leading to a complete self-consistent account of the molecular composition of vesicular stomatitis virus. The masses obtained were 265.6 +/- 13.3 megadaltons (MDa) for the native virion, 197.5 +/- 8.4 MDa for the pronase-treated virion, and 69.4 +/- 4.9 MDa for the nucleocapsid. The reduction in mass effected by pronase treatment, which corresponds to excision of the external domains (spikes) of G protein, leads to an average of 1,205 molecules of G protein per virion. The nucleocapsid mass, after compensation for the RNA (3.7 MDa) and residual amounts of other proteins, yielded a complement of 1,258 copies of N protein. Calibration of the amounts of M, NS, and L proteins relative to N protein by biochemical quantitation yielded values of 1,826, 466, and 50 molecules, respectively, per virion. Assuming that the remaining virion mass is contributed by lipids in the viral envelope, we obtained a value of 56.1 MDa for its lipid content. In addition, four different electron microscopy procedures were applied to determine the nucleocapsid length, which we conclude to be 3.5 to 3.7 micron. The nucleocapsid comprises a strand of repeating units which have a center-to-center spacing of 3.3 nm as measured along the middle of the strand. We show that these repeating units represent monomers of N protein, each of which is associated with 9 +/- 1 bases of single-stranded RNA. From scanning transmission electron microscopy images of negatively stained nucleocapsids, we inferred that N protein has a wedge-shaped, bilobed structure with dimensions of approximately 9.0 nm (length), approximately 5.0 nm (depth), and approximately 3.3 nm (width, at the midpoint of its long axis). In the coiled configuration of the in situ nucleocapsid, the long axis of N protein is directed radially, and its depth corresponds to the pitch of the nucleocapsid helix.

Capsid↗
PubMed1982

Histogenetic classification of lung carcinomas. small cell carcinomas studied by light and electron microscopy.

Forty-six small cell carcinomas of the lung identified by light microscopy were also studied by electron microscopy in an attempt to subclassify them histogenetically. Thirty were subclassifiable into neuroendocrine secretory, mucus secretory, or epidermoid categories on the basis of their differentiated structural features; 16 were too poorly differentiated to assign to any specific cell type and were designated null. In addition to histogenetic separation, electron microscopy revealed a spectrum of average cell differentiation within each subclass except null. The widest spectrum occurred in the most frequent subclass, the neuroendocrine group. This study clearly demonstrated that the light microscopic diagnosis of small cell carcinoma does not identify a morphologically homogeneous process. Whether histogenetic separation and/or some assessment of the relative degree of differentiation have any consistent clinical value remains to be shown; however, combined light and electron microscopy should improve the precision of the initial diagnosis over that of light microscopy alone.

Carcinoid Tumor↗
PubMed1984

Uses and contributions of diagnostic electron microscopy in surgical pathology: a study of 20 Veterans Administration hospitals.

Although the capability of electron microscopy to contribute to histopathologic diagnosis in selected instances, e.g., renal biopsies and biopsies of certain tumors, is generally recognized, its contribution in a broader range of surgical pathology accessions is not well established. To gain further information concerning this aspect of the use of electron microscopy, 7,193 surgical pathology reports, 290 (4 per cent) of which included electron microscopic findings, were analyzed; these reports represented all such accessions during the same single month at 20 VA medical centers. It was found that malignancies constituted 62 per cent of the lesions examined by electron microscopy but only 20 per cent of the total number of surgical cases examined by light microscopy. It was also found that the distribution of specimens by tissue site of origin differed considerably for specimens examined by electron microscopy and light microscopy; positive contributions of electron microscopy to diagnosis were found in 146 of the 290 electron microscopy cases (50 per cent), with higher percentages found in malignancies and in certain tissue site subsets (lower respiratory tract, soft tissue, kidney, skin, and lymph nodes with metastases); failure to examine some specimens (lung metastases to lymph nodes or other tissues and endobronchial biopsy specimens) suggests slight, but common, underutilization of electron microscopy in diagnosis.

Adult↗
PubMedDate not supplied

Pancreatic insulo-acinar portal systems in humans, rats, and some other mammals: scanning electron microscopy of vascular casts.

Scanning electron microscopy of vascular casts showed that in the mouse, rat, and guinea pig, the pancreatic endocrine islets were frequently interlobular in position and emitted insulo-venous efferent vessels directly draining into veins. In these animals, the intralobular islets, located within the exocrine lobules, issued insulo-acinar portal vessels continuous with the lobular capillaries in addition to the insulo-venous efferent vessels. In humans, monkeys, cows, pigs, dogs, cats, and rabbits, essentially all islets in the pancreas were intralobular in location and emitted the insulo-acinar portal vessels only. In man and animals examined, especially in the murine species, many lobules lacked an islet, therefore the insular control over the exocrine pancreas seemed to be effected in more or less restricted areas of lobules.

Animals↗
PubMed1992

Scanning electron microscopy of body fluids.

Scanning Electron Microscopic (SEM) examination of body fluid specimens submitted for cytopathological diagnosis is technically easy and can be accomplished within hours. Specimens refrigerated for as long as 72 hr show no significant structural alterations that would interfere with recognition of diagnostically significant cells. SEM is particularly well suited for these specimens because the cells are free floating, and even cells in clusters have "natural" surfaces. Clinical uses of SEM on body fluids include the identification of ambiguous cells, distinction of reactive (benign) mesothelial cells from metastatic adenocarcinoma, and distinction of lymphoid cells from small cell carcinoma of the lung. In addition, SEM of these fluids assists cytologists to better understand the cellular features and associations seen in the light microscope. Ultrastructural analysis can be an important component of the Quality Assurance Plan for those cytodiagnostic laboratories that have access to an electron microscopy facility.

Body Fluids↗
PubMed1992

Differences in ceramic-bone interface between surface-active ceramics and resorbable ceramics: a study by scanning and transmission electron microscopy.

The interface between bioactive ceramics and bone was studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The materials were apatite-wollastonite-containing glass ceramic (A-W.GC) as a representative surface-active ceramic, and calcite and beta-tricalcium phosphate (beta-TCP) as resorbable ceramics. Particles of these materials, ranging between about 100 microns and 300 microns in diameter, were implanted into rat tibiae, and specimens were prepared for observation at 8 weeks after implantation. Both SEM and TEM demonstrated that A-W.GC was bonded to bone through a thin Ca-P-rich layer consisting of fine apatite crystals apparently different from those of bone in shape, size, and orientation. Collagen fibers of the bone reached the surface of this layer, and chemical bonding between A-W.GC and the bone was speculated. Calcite and beta-TCP, on the other hand, made direct contact with the bone, and no apatite layer was present at the interface. The surfaces of the implants became rough due to degradation, and bone grew into the finest surface irregularities. However, we were unable to demonstrate any continuity of crystals between the resorbable implants and bone by high-resolution TEM. Accordingly, the bonding strength was considered to be mainly attributable to mechanical interlocking.

Animals↗
PubMed1983

Circulatory pathways in the sinusal spleen of the dog, studied by scanning electron microscopy of microcorrosion casts.

Scanning electron microscopy of microcorrosion casts was used to visualize circulatory pathways in the sinusal spleen of dog. The examination of contracted versus dilated organs and variations of the volume of material injected gave an indication of flow dynamics. Minimal injections of material into contracted spleens produced filling of mainly the fastest routes for flow, whereas injections into dilated spleens primarily filled slower routes. This procedure yielded a more complete, three-dimensional picture of the arterial, intermediate, and venous pathways as a whole, and of the relative amounts of flow through different arterial routes. Evidence of flow from capillary lumina out into ellipsoid sheaths was plentiful in casts from dilated spleens, but rare in casts from contracted organs. The pattern of flow within and out of the marginal sinus has been elucidated: A circumferential filling occurs first, followed by a flow that radiates outward into the marginal zone and red pulp. Venous sinuses filled via two routes in addition to the generally accepted path from the reticular meshwork via fenestrations in sinus walls. First, many venous sinuses extending out from the marginal sinus and surrounding marginal zone originated as open-ended tubes continuous with the reticular spaces of the marginal sinus or marginal zone. Second, direct connections of arterial capillaries with venous sinuses in the red pulp were found. Evidence indicating that some mechanism is controlling the flow via these routes is discussed. The strikingly different arrangement of venous sinuses in the subcapsular region is demonstrated.

Animals↗
PubMed1983

The intermediate circulation in the nonsinusal spleen of the cat, studied by scanning electron microscopy of microcorrosion casts.

Scanning electron microscopy of microcorrosion casts was used to visualize circulatory pathways of the intermediate circulation in nonsinusal spleen of cat. The marginal sinus (MS) around lymphatic nodules is a distinct vascular space which fills preferentially before the filling of the marginal zone (MZ) and surrounding red pulp occurs. The MS, which has a plentiful vascular supply, does not usually enclose the nodule completely. From the MS, flow occurs radially outwards into the MZ. Corrosion casts and histological sections both showed that a diversity of forms of the MZ exists: The thickness of MZ and the arrangement of its reticulum vary among nodules and between different areas of the same nodule, from a complete absence to a region of up to 50 microns in width. No direct arteriovenous connections were found (in contrast to dog spleen: Schmidt et al., '83b). Aside from capillary endings in the MS and MZ, all arterial capillaries terminate in the reticular spaces of the red pulp, i.e., the circulation appears to be entirely "open." From each capillary termination a great variety of flow pathways through the reticular meshwork to the pulp venules is available; some of these routes are quite long but others may involve distances as short as 15-25 microns. Evidence of flow into ellipsoid sheaths was abundant in casts from dilated spleens, but scarce in contracted spleens. In contrast to the extensive system of interconnected venous sinuses in dog spleen, the pulp venules found in cat spleen are nonanastomosing, shorter, and much smaller in caliber, and all receive flow freely from the reticular meshwork via open ends and fenestrations in their walls.

Animals↗
PubMedDate not supplied

Studies of the structural organization of a bacterial chemoreceptor by electron microscopy.

We used analysis by electron microscopy to obtain structural information about a transmembrane receptor that mediates chemotaxis in Escherichia coli. Two-dimensional arrays of regularly packed particles of the receptor Trg were obtained by reconstitution of purified, detergent-solubilized protein into lipid bilayers. Preliminary image processing of negatively stained arrays revealed an almost square 8.8 x 8.8-nm unit cell and resolved the particles into four peaks of density around a central depression. In certain conditions, reconstituted, Trg-containing bilayers associated into membrane stacks. The regular spacing of the stacks provided a value of 15 nm for the dimension of the receptor normal to the membrane. Using these dimensions, the estimated occupied volume of the structure would be sufficient to contain four monomers of Trg. This tetramer form may be a dimer of two antiparallel or parallel homodimers. Our analysis indicates that a receptor monomer is approximately 4.4 nm at the widest point and 15 nm long. Given the dimensions of the periplasmic domain of the closely related receptor Tars, determined by X-ray crystallography, and a minimum bilayer thickness of 3 nm, the cytoplasmic domain would be approximately 5.0 by 4.4 nm. Higher resolution analysis should reveal additional information about receptor structure.

Bacterial Proteins↗
PubMed1978

Nigrothalamic pathway in the cat demonstrated by autoradiography and electron microscopy.

Light microscopic autoradiography and electron microscopy were used to trace the nigrothalamic projections and to study the sites of termination of this pathway in the cat. Injections of tritiated amino acids or electrolytic lesions were placed in the substantia nigra pars reticularis (SNr). An accumulation of radioactivity was found in the ventral medial nucleus and in the ventromedial part of the ventral anterior nucleus. At the ultrastructural level degenerating medium size synaptic boutons and medium size myelinated fibers were observed in these nuclei. The boutons contained clear pleomorphic veiscles and formed symmetrical type synaptic contacts with regular type dendrites and vesicel-containing dendrites. The present findings indicate that the ventral medial nucleus is the principal site of termination of nigrothalamic projections in the cat.

Animals↗
PubMed1987

Complexes of the single-stranded DNA-binding protein from Escherichia coli (Eco SSB) with poly(dT). An investigation of their structure and internal dynamics by means of electron microscopy and NMR.

Based on electron microscopy and NMR spectroscopy it is deduced that Eco SSB binds with moderate cooperativity to polynucleotides. Evidence is provided that the protein binds in its tetrameric form to the nucleic acid forming a nucleosome-like structure. NMR-spectroscopic analysis of the complexes shows that the carboxy-terminal region of the Eco SSB maintains a high flexibility even when the protein is immobilized in large protein-protein clusters.

Escherichia coli↗
PubMed1986

Elemental imaging and resolution in energy-filtered conventional electron microscopy.

Energy-filtered transmission electron microscopy (EFEM) was used to image the distributions of uranium and carbon in uranyl acetate stained catalase crystals. The spatial resolution obtained from inelastic C K-edge and U O4,5-edge images, determined from the highest-order reflection in the computed diffraction pattern, was 3.4 nm for both carbon and uranium. The resolution limit imposed by the delocalization of inelastic scattering was estimated from cross-section measurements to be 0.6 nm for U and 0.2 nm for C. Considering both delocalization and the effects of microscope aberrations, for an objective lens chromatic aberration coefficient of 2.8 mm and 10 eV energy window, the calculated resolutions are 2.0 nm for C and 1.2 nm for U. The effects of plural inelastic and elastic-inelastic scattering were sufficiently large to show crystalline structure in unprocessed pre-edge inelastic images. Previously suggested methods for eliminating these artifacts were applied to obtain the compositional information in the catalase EFEM images.

Carbon↗
PubMedDate not supplied

Image analysis of Artemia salina ribosomes by scanning transmission electron microscopy.

A dedicated scanning transmission electron microscope (STEM) at Brookhaven National Laboratory was used to visualize unstained freeze-dried ribosomal particles under conditions which considerably reduce the specimen distortion inherent in the heavy metal staining and air-drying preparative steps used in routine transmission electron microscopy (TEM). From high-resolution STEM images it is possible to determine molecular mass and the mass distribution within individual ribosomal particles and perform statistical evaluation of the data. Analysis of digitized STEM images of Artemia salina ribosomes provided evidence that a standard preparation of these eukaryotic ribosomes consists of a population of heterogenous particles. Because of the integrity of rRNAs established by agarose gel electrophoresis, variations in the composition and structure of the 80S monosomes and the large (60S) and small (40S) ribosomal subunits, as monitored by their mass, were attributed to the loss of ribosomal proteins, from the large subunits in particular. These results are relevant not only to the degree of ribosomal biological activity, but should also be taken into consideration for particle selection in the reconstruction of the "native" eukaryotic ribosome 3-D model.

Animals↗