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Musculoskeletal responses of 2-year-old Thoroughbred horses to early training. 8. Quantitative back-scattered electron scanning electron microscopy and confocal fluorescence microscopy of the epiphysis of the third metacarpal bone.

AIM: To characterise and explain the increase in density evident by computerised tomography (CT) and radiography in companion studies as a response to training, in bone in the palmar and dorsal regions of the condyles of the third metacarpal bone (Mc3) of 2-year-old Thoroughbred horses. METHODS: Compositional back-scattered electron (BSE) imaging in scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM) were conducted on polymethyl methacrylate (PMMA)-embedded mediolateral slices of the right distal Mc3 from seven 2-year-old Thoroughbred horses trained on a racetrack and seven untrained horses kept at pasture. One left Mc3 from each group was studied in transverse section planes. This study focussed on regions of Mc3 found to differ in density between the trained and untrained horses in companion studies using CT and radiography. RESULTS: The increase of bone density in the condyles of Mc3 in trained horses compared with untrained horses occurred, without prior osteoclastic resorption, via the deposition of new bone on pre-existing internal surfaces. Within prior marrow spaces of cancellous bone, there was also rapid formation of immature strands and fronds of bone which were more cellular and mineralised, and more lamellar bone tissue was deposited on these new scaffolding elements in the trained horses. Both resulted in increased bone volume fraction (BVF). The microscopic mineralisation density of the bulk of the new tissue was lower than in pre-existing bone, and CT and radiography underestimated the increase in BVF. The new tissue was thus probably less stiff at the microscopic scale than pre-existing bone, though its addition would stiffen the global structure. CONCLUSIONS: In Mc3 of all the trained horses, there were obvious differences in microscopic structure compared with those from the untrained horses. Moderate, industry-standard levels of exercise used to prepare young horses for racing induced the formation of new bone in non-bone spaces in bone tissue, such that the bone organ should better withstand later increased levels of exercise.

Animals↗

A technique for quick fixation of whole Drosophila and other small insects for scanning electron microscopy.

A procedure for fixing small insects in natural postures for scanning electron microscopy is reported. Anesthetized insects are partially restrained using a depression slide and a coverslip while preliminary fixation is carried out and wings and legs are positioned with a fine brush. Following this, fixation is completed and the insect is prepared for scanning electron microscopy by essentially standard procedures, which may include critical point drying. Figures illustrate, however, that critical point drying is not necessary for more rigid parts of the exoskeleton. Use of this procedure assures naturally disposed parts even when only a single specimen is available.

Animals↗

Morphology of human cells, carrier of measles virus, using nomarski optics and scanning electron microscopy.

A human cell line (Lu106) carrier of measles virus was studied in a Nomarski interference-contrast microscope (NICM) and a scanning electron microscope (SEM). The results from this were correlated with fine structure findings obtained from analyses made in the transmission electron microscope (TEM). In both the NICM and SEM it was possible to identify intracellular perinuclear structures, which most likely represent aggregates of measles nucleocapsids. These structures appeared in the NICM as opaque vesicles and in the SEM as bulges in the flattened cells. The SEM also proved to be used for determining cell surface characteristics specific for the carrier culture, which were lacking in uninfected Lu106 cells. In the carrier culture, there were vesiculated cells with bled-like polymorphic and ridged projections, and cells with webbed cytoplasmic extensions. Ridges and transverse striations observed on these cellular protrusions and on microvilli possibly denote oriented viral nucleocapsids at the cell membrane. Furthermore in the carrier cells, the microvilli were more heterogenous in length and diameter and were more frequently branched or fused together when compared to microvilli in uninfected cells. The results are discussed in view of the available information on the appearance of virus-infected or virus-transformed cells in the SEM, also inregard to the various factors, other than virus infection, which play a role in determining the surface features of monolayer cells.

Cell Line↗

Eimeria dispersa, E. adenoeides, and E. meleagrimitis: intestinal mucosal disruption in turkeys as seen with scanning electron microscopy.

Tissues from the digestive tract of turkeys infected with Eimeria dispersa, E. adenoeides, or E. meleagrimitis were compared with tissues from uninfected controls as seen with light microscopy (LM) and scanning electron microscopy (SEM). Six regions were examined--duodenum, jejunum, ileum, cecal neck, cecal pouch, and large intestine. Although LM showed large numbers of E. dispersa in the epithelial cells, SEM usually showed little mucosal disruption. Occasionally the surface of duodenum and jejunum, as seen with SEM, was convoluted and disrupted. In one bird, some parasite-induced damage was found with either LM or SEM in the duodenum and jejunum of turkeys given E. adenoeides oocysts. Although LM showed parasites in the rest of the digestive tract of all E. adenoeides infected birds, SEM showed only a localized sloughing of the mucosa in the cecal pouch. The most extensive damage to the villar surface was caused by E. meleagrimitis. Infections often disrupted the villar tips, especially in the small intestine and cecal neck. Localized areas of pitting were often found on individual villi. All 3 species produced oocyst extrusion sites, especially in the ileum and the ceca.

Animals↗

An improved and simplified method for combined light and scanning electron microscopy in human urinary cytology.

Human bladder-washing and voided urine specimens from normal cases and inflammatory and malignant lesions were examined by the combined use of light microscopy (LM) and scanning electron microscopy (SEM). A newly-designed mesh, which consisted of a piece of gelatin-covered, osmium-impregnated and poly-L-lysine-coated glass slide, 0.85 mm in thickness and containing 42 compartments of 25 sq mm in size, was used in this study. This mesh permitted a direct correlation of LM and SEM images, thus shortening the time of observation of specimens under SEM. With SEM, the diagnosis of malignant exfoliated urothelial cells depended on the presence or absence of pleomorphic microvilli. The technique has enhanced the accuracy of diagnosis over conventional methods, especially for noninvasive, low-grade, papillary tumors of the urinary bladder.

Humans↗

Scanning electron microscopy of experimentally induced sequential alterations of rat liver hyperplastic nodules.

Rat liver hyperplastic foci (HF) and hyperplastic nodules (HN) induced by diethylnitrosamine (DEN) according to the method of Solt and Farber were observed by light microscopy (LM) and scanning electron microscopy (SEM). Liver cirrhosis was not observed. HF, which were composed of basophilic hepatocytes arranged usually in two-cell-thick plates and occasionally one-cell-thick plates, were followed by early HN, which were composed of eosinophilic hepatocytes with enlarged nuclei and prominent nucleoli, at 4 weeks after DEN administration. SEM revealed that hepatocytes in HF had smooth surfaces and dilated bile canaliculi with a few short microvilli. Early HN (4 and 6 weeks) were composed of two-cell-thick plates of hepatocytes, whose intercellular surfaces were covered with numerous microvillous projections. Bile canaliculi were dilated and had numerous short microvilli. Late HN (6 and 8 months) were composed of eosinophilic hepatocytes, which were irregular in shape and size. Their nuclei were enlarged and nucleoli were prominent. Hepatocytes were arranged in two-cell-thick plates, but never in many-cell-thick plates. Bile canaliculi were larger than sinusoids. The bile canaliculi had numerous diverticula, but the zona occludens was present. Microvillous projections of intercellular surfaces of hepatocytes were more numerous than those of early HN. Fenestrae of the sinusoidal endothelium were lost in most parts. Kupffer cells were not seen in any stage of HN. HN progressed gradually toward hepatocellular carcinoma.

Animals↗

Detection of early neoplastic changes in experimentally induced colorectal cancer using scanning electron microscopy and cell kinetic studies.

Colonic tumours were induced in Wistar rats using 12 consecutive subcutaneous injections of azoxymethane at a dose of 10 mg/kg/week. Pairs of rats were killed at five weekly intervals after initial injection until 25 weeks. Colonic mucosa was sampled from five standard areas along the length of the colon and examined by both scanning electron microscopy and conventional light microscopy. The crypt cell production rate was measured by stathmokinetic techniques. Scanning electron microscopy showed microadenomas as early as five weeks and consistently after 15 weeks. They were found predominantly in the distal colon and increased in size with time. The lesions showed a progressive increase in the number of crypts per adenoma and increasingly disorganised slit shaped crypt orifices. The presence of epithelial dysplasia in the microadenomas and of invasion of the colonic wall by carcinoma was confirmed histologically, although fewer lesions were identified in tissue sections than by scanning electron microscopy. Crypt cell production rate increased with time, particularly in the distal colon. This increase was significant between five and 25 weeks. The results of these observations suggest that there is an adenoma-carcinoma sequence in this animal model. The value of scanning electron microscopy in identifying and quantifying the mucosal changes during carcinogenesis is emphasised.

Adenoma↗

Scanning electron microscopy and profilometer evaluation of glazed and polished dental porcelain.

The surface roughness of Vita VMK porcelain following oven glazing and eight grinding/polishing treatments was analyzed qualitatively using scanning electron microscopy and quantitatively using a profilometer. Techniques were selected to simulate clinical practice. Scanning electron microscopy evaluation found oven glazing produced a better surface than other polishing methods. On the basis of the profilometric examination, the best roughness average value was obtained using diamond instruments with progressively smaller particle sizes (30, 15, and 8 microns). Scanning electron microscopy analysis showed that all the treatments left the surfaces partially porous and cracked; however, the glazed surface yielded the best result. Although no significant differences were detected for the different treatments, the use of a 30-microns diamond instrument produced a rougher surface. No correlation was found between scanning electron microscopy and profilometer results. This study suggests that several procedures may be used to effectively finish ceramic surfaces.

Analysis of Variance↗

Scanning electron microscopy substantiates histology in showing the inadequacy of the existing theories on the development of the proximal coronary arteries and their connections with the arterial trunks.

Development of proximal coronary arterial segments and coronary arterial orifices was studied by scanning electron microscopy in 20 rat embryos and by light microscopy in serial sections of 20 human and another 18 rat embryos. Neither by scanning electron microscopy nor by light microscopy did we observe more than two coronary arterial orifices. These coronary orifices were always situated in the sinuses of the aorta that faced the pulmonary artery. In the human embryos the coronary orifices emerged between 37-39 days of gestation (16-19 mm crown-rump length, Streeter horizon XVIII-XIX) and were invariably present beyond 39 days (19 mm crown-rump length, Streeter horizon XIX). In rat embryos, the coronary orifices emerged in both scanning electron microscopy and light microscopy at 15-17 days of gestation (13-17 mm crown-rump length) and were invariably present beyond 17 days (17 mm crown-rump length). In both human and rat embryos, either by scanning electron microscopy and light microscopy, the left coronary orifice was observed significantly earlier. In all the investigated embryos, human as well as rat, septation at arterial orifice level was complete, including the earliest stages studied. Light microscopy showed that at the emerging stages of the coronary orifices, the proximal epicardial segments of the left and right coronary arteries could already be identified in a peritruncal ring of epicardial vasculature, before the coronary orifice was observed. This was the case in human as well as in rat embryos. Thus, a coronary orifice was never seen in the absence of a proximal coronary artery. The present theories on development of the proximal coronary arteries and coronary orifices do not offer an adequate explanation for either these data or the known possible congenital abnormalities of the coronary arteries. Our study supports dual proximal coronary arterial development. These two proximal coronary arteries develop out of a peritruncal ring of vascular structures on to the aorta. The process by which the coronary orifices actually develop remains to be explained.

Animals↗

Scanning electron microscopy study of articular cartilage in the evolution of hip arthritis.

The authors report the results of scanning electron microscopy examination of samples of articular surface taken from 15 arthritic femoral heads removed during hip replacement. The specimens were collected from areas of the femoral head where different degrees of degenerative changes were identified, and processed with the normal techniques for scanning electron microscopy. A definition was made of the mechanisms leading to articular cartilage wear, the most obvious sign of cartilage degeneration. The earlier stages were characterized by the appearance of clefts in the surface layer, which merged and formed scales that flaked off, giving rise to superficial abrasions. The evolution of the degenerative process led to a deepening of the clefts, reaching the intermediate and deep layers and creating further scales of cartilage tissue, oblique or perpendicular to the surface. The elimination of these formations causes more or less marked wear that can extend down to the subchondral bone (ulcers and ground surfaces). As well as information on the condition of the articular surfaces, scanning electron microscopy may also provide details on the morphology and structure of the chondrocytes and the fibrillar network in the sagittal sections, showing the regressive phenomena associated with arthritis.

Aged↗

Light and scanning electron microscopy of exfoliated bladder epithelial cells in rats fed N-(4-(5-nitro-2-furyl)-2-thiazolyl)formamide.

Urine specimens obtained periodically from weanling male F344 rats fed 0.2% N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) for 25 weeks followed by control diet for an additional 35 weeks, were examined by light microscopy (LM) and scanning electron microscopy (SEM). LM revealed red blood cells and atypical transitional cells in the urine at 25 and 43 weeks, when microinvasive transitional cell carcinoma was present. Malignant transitional cells were seen in the urine by LM at 60 weeks, when muscle invasion was present in histologic sections. However, SEM showed pleomorphic microvilli on exfoliated urothelial cells as early as 10 weeks after the beginning of FANFT administration, and the microvilli became increasingly pleomorphic as the bladder lesions progressed to muscle invasion at 60 weeks. Noninvasive tumors were present in histologic sections of rat bladders examined at the end of 10 weeks of FANFT administration. The numerous microvilli on exfoliated bladder tumor cells were in sharp contrast to the peaked microridges of normal exfoliated superficial cells.

Animals↗

Scanning electron microscopy of brain ventricular surfaces: a bibliography.

This bibliography is designed to facilitate the rapid identification and location of references concerning the application of scanning electron microscopy to investigations of the cerebroventricular system and related cerebrospinal fluid contacting surfaces. Because SEM provides information regarding cell surfaces, it is admirably suited to the examination of the brain ventricular system since the morphology of the CSF interface is of the utmost importance in accomplishing the diverse functions of the ependyma and associated circumventricular organs. The bibliography attests to the fact that scanning electron microscopy has provided a major impetus to the study of the structure and function of the circumventricular organs, choroid plexuses and tanycytic ependyma as well as the distribution and morphology of such supraependymal cellular elements as macrophages, neurons, and neuronal proceses. Furthermore, not only are SEM methodologies being applied to an ever lengthening list of phyla and species, but the diversity of problems amenable to the profitable application of scanning electron microscopy also is rapidly increasing. Investigations to date, for example, include studies of ventricular morphology during development and maturity, health and disease, and prior to as well as following experimental manipulation. While remarkable strides have been made toward a greater understanding of many aspects of cerebroventricula structrure and function, many significant problems clearly await the judicious application of scanning electron microscopy.

Bibliographies as Topic↗

Scanning electron microscopy of the human myocardium.

Owing to the impossibility of fixation, in the human being, by means of perfusion in vivo and the rare availability of fresh comparatively intact hearts resected at transplantation, the possibility was explored of using post-mortem material in instances where the autopsies were performed within 2 hours of demise of the patients. Four cases were examined: 2 normal adult hearts and 2 hearts of hypertensive patients in the stage of decompensation. The hearts were fixed by means of perfusion with 2.5% glutaraldehyde in phosphate buffer, pH 7.4. Contiguous blocks were taken for light microscopy (LM) and scanning electron microscopy (SEM) from the left ventricular free wall of all 4 cases and of the impulse conducting system of the heart in the 2 normal hearts. The SEM material was processed by the osmium-thiocarbohydrazide-osmium method (O-T-O). There was good correlation between the LM and SEM findings. The left ventricular blocks were sectioned in the transverse axis and SEM showed a step-wise transection of the myofibrils. Z and M bands, mitochondria and myofilaments were identified a ultrastructural magnifications. The difference in vascularity between normal and hypertensive myocardium and the presence of para-arterial fibrosis in the latter were demonstrated by SEM. The SEM study of the impulse conducting system of the heart included the sinoatrial (SA) and atrioventricular (AV) nodes and the penetrating portion of the atrioventricular (AV) bundle of His. Characteristic pacemaker or "P" cells were identified in the nodes.

Adult↗

A new method for isolation of leukocytes from the peripheral blood of amphibians [Bufo himalayanus, (Gunther)] and study of their surface morphology by scanning electron microscopy.

Using percoll as the density gradient, a new single step method to isolate leukocytes from the peripheral blood of amphibians (B. himalayanus) has been described. Isolated leukocytes were photographed under the scanning electron microscope and an attempt has been made to characterize the leukocyte population on the basis of surface morphology. Apart from regular blood cell types, B. himalayanus have slender, elongated and slightly curved leukocyte type cells in their peripheral blood. Such slender elongated cells were absent in the blood of a related species B. stomnaticus and hence could not be categorized under the known blood cell types.

Animals↗

[Interaction of silicone oil with various intraocular lenses. A light and scanning electron microscopy study].

BACKGROUND: Silicone oil is increasingly used as an intravitreal tamponade in severe forms of vitreoretinal disorders, also in pseudophakic eyes. In some patients silicone oil was observed to be adherent to the intraocular implant. MATERIAL AND METHODS: To investigate the interaction of silicone oil with IOL-materials and the influence of lipoproteins of the serum on this interaction we used the following in vitro experimental set-up: Sterile IOLs of different IOL-materials (e.g. PMMA, silicone, hydrogel) and surface-modifications (e.g. Pharmacia 809P and 809C, Soflex LI41U, Alcon AcrySof M60BM, Allergan SI-30NB, SI-40NB, Chiron adatomed 90D, C10 and C31UB, MemoryLens U940A, Corneal ISHEMA 66, Storz H60M) were stored in vitro together with silicone oil (1000 centistokes) with and without a fresh serum solution at 37 degrees Celsius. This combination was rotated slowly and mixed thoroughly three times a day. After 3 and 6 months and a standardized special preparation in an ultrasound bath and air treatment, the IOLs were examined by light microscopy followed by scanning electron microscopy. RESULTS: Silicone oil strongly adhered to holes and to the IOL surface especially in silicone and PMMA IOLs. Highly hydrophylic IOLs-like the heparin-surface-modified and hydrogel IOL-cleared from silicone oil relatively early. This effect was more pronounced in the group with lipid-/serum solution than without. In some silicone IOLs silicone oil was strongly adherent to the lens and it was nearly impossible to wash off the oil even when using an alcoholic solution. One silicone IOL lost its haptics in the serum-oil-IOL-mixture, another lost small particles from the IOL surface. CONCLUSION: Silicone oil is more adherent to hydrophobic IOLs than to hydrophylic IOLs. Lipoproteins of the serum play an important role in facilitating an effect of silicone oil on IOL materials. We recommend highly hydrophylic especially surface-modified IOLs for lens implantation following vitreoretinal silicone oil surgery. Implantation of silicone IOLs in vitreoretinal high risk eyes should be avoided.

Equipment Failure Analysis↗

Scanning electron microscopy of vertebrate cerebellar cortex.

In this study, the Golgi method for light microscopy, transmission and conventional scanning electron microscopy, the ethanol-cryofracturing technique, the freeze-fracture method for SEM, and the freeze-etching method have been used in conjuction to analyze the three-dimensional cytoarchitectonic arrangement and intracortical circuits of vertebrate cerebellar cortex. Approximately more than 100 specimens of mice, rat, teleost fishes and human cerebelli were processed by the above mentioned techniques. A chronological review of other methods for studying hidden surfaces of cerebellar nerve cell has been also described. The three-dimensional morphology, outer and inner surfaces of granule, Golgi, Purkinje and stellate cells were reviewed by means of a correlative and comparative study. The cerebellar circuits formed by mossy and climbing fibers with granule cell dendrites and Purkinje cell-granule cell synapses have been traced by ethanol-cryofracturing technique, freeze-fracture method for SEM and freeze-etching technique. These findings have been correlated with previous light and electron microscope findings published in the last century. Some advantages and limitations of each method are pointed out. The review emphasizes the paramount importance of correlating light microscope Golgi method with ethanol-cryofracturing and slicing techniques for SEM. The correlation between freeze-fracture method for SEM and freeze-etching technique provides a new approach for studying three-dimensional morphology of nerve cells at cellular and macromolecular levels. This modern methodology of three-dimensional analysis offers new potential areas for future experimental investigation in embryology and pathology of the central nervous system.

Animals↗

Scanning electron microscopy of Barrett's epithelium and its correlation with light microscopy and mucin stains.

The surface epithelial cells of Barrett's esophagus were characterized using quantitative scanning electron microscopy and light microscopy with mucin histochemical stains. Fifty-one biopsy specimens of Barrett's esophagus from 15 patients and 31 control specimens of the stomach and intestines from 9 patients were examined. Three distinct surface cell types, in addition to the goblet cell, were recognized in Barrett's epithelium: the gastric-like cell in 31% of specimens, which was similar to the normal gastric surface cell by quantitative scanning electron microscopy; the intestinal-like cell in 41%, which was most similar to the normal small intestinal surface cell; and the variant cell in 80%, which had a range of surface features. By light microscopy, all specimens with variant and intestinal-like cells were classified as specialized columnar epithelium. The surface mucous cells in Barrett's epithelium displayed a variety of mucin staining patterns with acid nonsulfated (small intestinal-like) mucin present in 90% of specimens and acid sulfated (colonic-like) mucin in 43% of specimens. Quantitative scanning electron microscopy and mucin histochemical stains reveal a striking cellular heterogeneity not apparent by routine light microscopy.

Barrett Esophagus↗

Scanning electron microscopy of Giemsa-stained chromosomes and surface-spread chromosomes.

Giemsa-stained chromosomes as prepared for light microscopy, and including G-banded, C-banded, and FPG-stained chromosomes, were examined by scanning electron microscopy. Although suitable for light microscopy, these chromosomes were too flat for a close examination of their fine structure by scanning electron microscopy. The surface of Giemsa-positive regions was rough and bright, whereas that of unstained or poorly stained regions was smoother and less bright. Giemsa-staining, therefore, seems to produce the bulkiness of the chromosomes. On topographical examination by scanning electron microscopy, the transparent chromosomes as observed with the light microscope proved to be "footprints". Stereographical examinations of surface-spread chromosomes showed that minimally stretched chromosomes were composed of a mass of nodular and twisted looping fibers with an average diameter of about 300 A. The substructure of these chromosome fibers was not determined. The kinetochore region was discernible as a constriction in the mass of the chromosome fibers, and was distinguishable from gaps by the presence of several chromosome fibers parallel to the axis of the chromatid. The organization of the chromosome fibers, however, was disordered rather than regular.

Azure Stains↗