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The bending of sliding microtubules imaged by confocal light microscopy and negative stain electron microscopy.

Individual microtubules can be visualised by confocal microscopy in reflection mode; when associated with a glass surface, they show up as black lines against the bright reflection from the surface. The high contrast imaging allows details of the behaviour of sliding microtubules to be studied easily. Taxol-stabilised microtubules sliding over kinesin-coated surfaces are normally straight, but can bend into tight loops if the leading end sticks to the surface. Some remain curved after release and move in circles. In such cases, the microtubule lattice must have become stably deformed. Electron microscopy of microtubules fixed during sliding shows no gross rearrangement of the subunit lattice and indicates that microtubule bending is mainly achieved by increased twisting of the longitudinal protofilaments around the microtubule.

Adenosine Triphosphatases↗

Topographic examination of sister chromatid differential staining by Nomarski interference microscopy and scanning electron microscopy.

BrdU-substituted Chinese hamster chromosomes were treated with a hog Na2HPO4 solution and stained with Giemsa to produce sister chromatid differential staining (SCD). The process of SCD was examined with the Nomarski differential interference microscope and the scanning electron microscope. After the Na2HPO4 treatment alone, unifilarly BrdU-substituted (TB) chromatids appeared somewhat more severely collapsed than the bifilarly substituted (BB) chromatids. Subsequent Giemsa staining, however, brought about pronounced piling up of the Giemsa dye on the TB-chromatids but not on the BB-ones, causing highly distinct differential Giemsa staining as well as a marked differentiation in surface topography between the sister chromatids. Removal of the Giemsa dye from the differentially Giemsa stained chromosomes resulted in a disappearance of such a pronounced topographic differentiation.

Animals↗

A novel sample holder allowing atomic force microscopy on transmission electron microscopy specimen grids: repetitive, direct correlation between AFM and TEM images.

A novel sample holder that allows atomic force microscopy (AFM) to be performed on transmission electron microscope (TEM) grids is described. Consequently, AFM and TEM images were repeatedly obtained on exactly the same sample area. For both techniques, a thin carbon film was used as the imaging substrate. Although these techniques have been previously used in conjunction, AFM and TEM images on exactly the same area have not been repeatedly obtained for any system. Correlation of AFM and TEM images is useful for work where the three-dimensional topographical information provided by the AFM could be used to better interpret the two-dimensional images provided by the TEM and vice versa. To demonstrate the applicability of such correlation, new results pertaining to a fibrillar collagen system are summarized.

Fibrillar Collagens↗

One to one correlation of histological and histochemical light microscopy with scanning electron microscopy.

Several histological stains were applied to specimens after scanning electron microscopic (SEM) processing. Histochemical stains were applied before SEM fixation. After staining, the specimens were processed and dried by SEM techniques. The specimens were taped to a microslide, specimen side up, scribed and covered with immersion oil. After light micrography (LM), the oil was removed and the specimens mounted and gold coated. The same cells were then relocated and photographed by SEM. Periodic acid Schiff was not usable as a specific tissue aldehyde stain, but did prove to be a useful counterstain for dehydrogenase stained specimens. Colloidal iron, as seen by SEM, resulted in a non-specific granular deposit over the specimen and the substrate. All the other histological stains examined--alcian blue, Grams, Feulgen and toluidine blue--were specific and did not change the specimen ultrastructure. The histochemical stains--acid and alkaline phosphatase and three dehydrogenases--were also specific. There were some minor ultrastructural changes with the AcPase and AlPase stains. However, the tissue surface was not significantly distorted. These and other techniques should prove to be a useful adjunct to SEM studies.

Amniotic Fluid↗

Salmonella 3, 10:r: toxicity in rabbit ileum and liver by light and electron microscopy.

Salmonella 3, 10:r:- (a monophasic variety of otherwise diphasic serotypes such as S. weltevreden and S. simi) Cell_free filtrate, when introduded into rabbit ligated ileal loops causes fluid exsorption, as studied 18-hr after treatment. Light microscopic histology of treated ileum shows denudation of the columnar epithelium at several places, thereby allowing the passage of the toxic principle into circulation. An important target organ, liver shows extensive centrilobular necrosis, as observed by light microscopy. Transmission electron microscopy of ileum reveals opening of membrane junctions between the adjacent cells of epithelial lining of the treated ileum at places, and focal devitalization including formation of intra-cellular membranous inclusinos. Electron microscopy of liver shows extensive damage and swelling of cytoplasmic membranes. However, the areas of darkly staining lamellae of granulated endoplasmic reticulum are also seen in stacks as will as dispersed. These studies stress that Salmonella toxic substances can cause extensive damage to intestine and liver both.

Animals↗

Effect of dopamine agonist medication on prolactin producing pituitary adenomas. A morphological study including immunocytochemistry, electron microscopy and in situ hybridization.

Conventional light microscopy, immunocytochemistry, electron microscopy and in situ hybridization were used to evaluate the effect of dopamine agonists (bromocriptine-LAR and bromocriptine) on the morphology of surgically removed prolactin (PRL)-producing pituitary adenomas. Dopamine agonist therapy resulted in decrease of serum PRL, clinical improvement and tumour shrinkage. Using light and electron microscopy cellular atrophy, interstitial and perivascular fibrosis were noted; in several tumours connective tissue accumulation was pronounced. The cellular response was not uniform. In some adenomas populations of large cells and small cells were distinguished. The large cells contained immunoreactive PRL and expressed the PRL gene indicating resistance to dopamine agonists. It appears that these cells retained the potential to secrete PRL and proliferate despite exposure to dopamine agonists. In the small cells, PRL immunoreactivity and PRL gene expression decreased providing evidence that both PRL release and synthesis were blocked. Small cells can persist in tumours after discontinuation of dopamine agonist medication suggesting these small cells are irreversibly suppressed and are not capable of regaining their endocrine function and proliferative capability. The formation of irreversibly suppressed PRL cells may explain why some PRL-producing adenomas do not recur after withdrawal of dopamine agonists.

Adenoma↗

Comparative analysis of the proliferative response of the rat urinary bladder to sodium saccharin by light and scanning electron microscopy and autoradiography.

Three methods used to detect proliferative changes in the rat urothelium, light microscopy, scanning electron microscopy, and autoradiography, were compared for their sensitivity in detecting changes produced by administration of sodium saccharin. Weanling male F344 rats were fed sodium saccharin as 0, 3, 5, or 7.5% of the diet, and the bladders were evaluated after 4, 7, and 10 wks of feeding. Light microscopic changes and an increase in labeling index were seen at all time points in rats fed 7.5% sodium saccharin, but not at the lower doses. A slight increase in labeling index was also observed at 10 wks in the 5.0% group. Scanning electron microscopic changes were evident as early as 4 wks with increasing severity at the 3, 5, and 7.5% doses. This study demonstrates that the hyperplastic response of the urothelium to sodium saccharin administration varies with dose and time, and that observation by scanning electron microscopy is the most sensitive of the three methods evaluated for detecting these changes.

Animals↗

Visualization of the cytostome in Trypanosoma cruzi by high resolution field emission scanning electron microscopy using secondary and backscattered electron imaging.

High resolution scanning electron microscopy was used to analyze the surface of epimastigote, amastigote and trypomastigote forms of Trypanosoma cruzi. Significant differences were observed between these forms and in different areas of the same cell. The cytostome found in amastigote and epimastigote forms could be easily visualized in images, which resemble those obtained only using the freeze-fracture technique. In contrast to other areas of the cell surface, the region of the cytostome, localized close to the flagellar pocket, showed a rugous surface and an opening with a diameter of 90 nm. Gold-labeled concanavalin A binds to the whole cell surface. However, the extent of binding was much higher in the region of the cytostome. The results obtained show that high resolution scanning electron microscopy is a powerful technique for analyzing the surface of protozoa.

Animals↗

Structural organization of an active, chromosomal nucleolar organizer region (NOR) identified by light microscopy, and subsequent TEM and STEM electron microscopy.

The three-dimensional arrangement of the chromatin components within the nucleolar organizer regions (NORs) from living oocyte nuclei was investigated. As a suitable cell system we chose vitellogenic oocytes of the orthopteran insect Acheta. This cell type is particularly attractive for analysis of nucleolar chromatin, since structural and functional aspects of NORs during early oogenic stages (including the association of NORs with amplified rDNA copies) are particularly well known (Lima-de-Faria 1974). In the course of the present study we first identified putative chromosomal NORs in isolated nuclei of mid-diplotene oocytes according to morphological characteristics using differential interference contrast (DIC) or phase contrast light microscopy. The presence of actively transcribing chromosomal NORs during this late stage of Acheta oogenesis obviously had been overlooked by previous investigators, probably due to difficulties of chromosome visualization. For a more detailed ultrastructural analysis, NORs were gently sedimented from opened nuclei and processed for sectioning using a modified "end-embedding" procedure (Mott and Callan 1975; Spring and Franke 1981). A small number of thick and thin sections could be made from individual NORs. Sections were analyzed by light microscopy, conventional transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). Whereas the structural connection of NORs to the chromosome axis and also the general arrangement of active nucleolar genes within the NOR complex could be seen with TEM, the visualization of individual nucleolar genes and the organization of transcription complexes was only possible using bright field STEM of thick sections at low temperature.

Animals↗

Endothelial cell density determined by specular microscopy and scanning electron microscopy.

Human eyes were photographed with a specular microscope and later examined wit a scanning electron microscope. Corneas from patients undergoing corneal transplantation in whom we were able to obtain preoperative specular micrographs were similarly analyzed. An attempt was made to correlate the counts obtained with both microscopic techniques by determining the amount of shrinkage the cornea undergoes while being processed for SEM. All specimens were counted with a planimeter. We found that the specular microscope adequately analyzes the endothelial cell density in the central and paracentral cornea of a normal eye, but because of its small sampling area specular microscopic counts are subject to significant error when dealing with nonhomogeneous populations such as postoperative cases. We found the peripheral corneal endothelial density to be less than the central endothelial density. Furthermore, we found that we could maximize the accuracy of counting by using a variable frame in a nonhomogeneous population, counting a minimum of four photographs per specimen, analyzing different areas, and analyzing larger areas.

Adolescent↗

Accessing nuclear structure for field emission, in lens, scanning electron microscopy (FEISEM).

Scanning electron microscopy (SEM) has had a shorter time course in biology than conventional transmission electron microscopy (TEM) but has nevertheless produced a wealth of images that have significantly complemented our perception of biological structure and function from TEM information. By its nature, SEM is a surface imaging technology, and its impact at the subcellular level has been restricted by the considerably reduced resolution in conventional SEM in comparison to TEM. This restriction has been removed by the recent advent of high-brightness sources used in lensfield emission instruments (FEISEM) which have produced resolution of around 1 nanometre, which is not usually a limiting figure for biological material. This communication reviews our findings in the use of FEISEM in the imaging of nuclear surfaces, then associated structures, such as nuclear pore complexes, and the relationships of these structures with cytoplasmic and nucleoplasmic elements. High resolution SEM allows the structurally orientated cell biologist to visualise, directly and in three dimensions, subcellular structure and its modulation with a view to understanding, its functional significance. Clearly, intracellular surfaces require separation from surrounding structural elements in vivo to allow surface imaging, and we review a combination of biochemical and mechanical isolation methods for nuclear surfaces.

Animals↗

The renal vascular lesions of severe and malignant hypertension: a light-immunofluorescent microscopy, transmission and scanning electron-microscopy study.

1. Arterial and glomerular lesions were studied in seventy-eight specimens from patients with severe or malignant primary or secondary hypertension by transmission and scanning electron microscopy and light-microscopy and immunofluorescent microscopy. 2. The lesions were classified as compensatory, decompensated and reparative. The arterial lesions of primary and secondary hypertension were qualitatively similar. Each of the microscopy techniques contributed to a better understanding of the changes.

Basement Membrane↗

The cellular transformation of injected colloidal iron complexes into ferritin and hemosiderin in experimental animals; a study with the aid of electron microscopy.

As revealed by electron microscopy and electron diffraction, the physical state of ferric hydroxide micelles contained in iron-dextran, saccharated iron oxide, and hydrous ferric oxide ("ferric hydroxide") differs notably from the state of the ferric hydroxide in ferritin or hemosiderin. By virtue of this difference one can trace the intracellular transformation of colloidal iron, administered parenterally, into ferritin and hemosiderin. One hour after intraperitoneal injection of iron-dextran or saccharated iron oxide into mice, characteristic deposits were present in splenic macrophages, in sinusoidal endothelial cells of spleen and liver, and in vascular endothelial cells of various renal capillaries. Four hours after injection, small numbers of ferritin molecules were identifiable about intracellular aggregates of injected iron compounds; and by the 6th day, ferritin was abundant in close proximity to deposits of injected iron compounds. The latter were frequently situated in cytoplasmic vesicles delimited by single membranes. These vesicles were most frequently found in tissue obtained during the first 6 days after injection; and in certain of the vesicles ferritin molecules surrounded closely packed aggregates of injected material. Much unchanged ferric hydroxide was still present in macrophages and vascular endothelial cells 3 to 4 weeks after injection. While electron microscopy left no doubt about the identity of injected ferric hydroxide on the one hand, and of ferritin or hemosiderin on the other, histochemical tests for iron failed in this respect. Precipitation of ferric hydroxide (hydrous ferric oxide) from stabilized colloidal dispersions of iron-dextran was brought about in vitro by incubation with minced mouse tissue (e.g. liver), but not by incubation with mouse serum or blood. Subcutaneous injections of hydrous gel of ferric oxide into mice initially produced localized extracellular precipitates. Most of the material was still extracellular 16 days after injection, though part of it was phagocytized by macrophages near the site of injection; but apparently none reached the spleen in unaltered form. Five days after injection and thereafter, much ferritin was present in macrophages about the site of injection and in the spleen. The findings show that iron preparations widely used in therapy can be identified within cells, and that their intracellular disposition and fate can be followed at the molecular level. Considered in the light of previous work, they indicate that the characteristic structure of the ferric hydroxide micelles in molecules of ferritin is specific, and develops during the union of apoferritin with ferric hydroxide. Apparently this union does not depend upon specific cell components.

Animals↗

Surface alterations in dialysis roller pump inserts: a scanning electron microscopy study.

Scanning electron microscopy has been used in a study of particle spalling from silicone and PVC elastomer inserts of dialysis roller pumps. In an experimental circuit using Isoton II, pump occlusion, pressure, pump revolutions and temperature were monitored. Scanning electron microscopy was performed on inserts exposed to 1-10 h of pumping using Silastic (medical grade) and a Watson-Marlow MHRE pump, with PVC and Pivipol, a polyurethane coated PVC (Bellco) using a Bellco BL705N pump. Changes included longitudinal splits (single and multiple), superficial cracks (random and orientated), deformations of normal surface pattern, holes, craters and detaching particles. There was an increase in these abnormalities with time for all types of pump insert, the changes being most marked for the Silastic/Watson-Marlow pump combination and least prevalent with the PVC/Bellco pump combination.

Humans↗