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Effect of pretreatment with ethanol containing fluoride on etched enamel surface as evaluated by scanning electron microscopy, optical microscopy, and tensile bond strength measurement.

The effects of pretreatment with a fluoride-containing ethanol solution on etched enamel surfaces before application of sealant were examined using scanning electron microscopy, optical microscopy, and tensile bond strength determinations. The results suggested that when etched enamel surfaces are treated with ethanol containing no more than 250 ppm fluorine in potassium fluoride, there is no interference with the bonding of the sealant to the treated, etched anamel surface.

Acid Etching, Dental↗

Monitoring morphology and signal during non-radioactive in situ hybridization procedures by reflection-contrast microscopy and transmission electron microscopy.

We analyzed the effects of steps in RNA in situ hybridization (ISH) procedures on morphology and hybridization signal with reflection-contrast microscopy (RCM) and transmission electron microscopy (TEM). In chessboard experiments, a range of fixatives containing formaldehyde, glutaraldehyde, or both, and various permeabilization protocols, including ethanol and pepsin treatment, were investigated. A transfected rat fibroblast cell line that harbors an inducible human cytomegalovirus immediate early (IE) transcription unit, and specific probes for 28S ribosomal RNA and IE messenger RNA were used for this purpose. Probes were labeled with digoxigenin and hybrids were detected with anti-digoxigenin F(ab)2 fragments conjugated to horseradish peroxidase, followed by diaminobenzidine/H2O2 reaction. Effects of fixation and pre-treatments on RNA detection efficiency and morphology were monitored by RCM on whole cells. After Epon embedding and ultra-thin cross-sectioning, the corresponding TEM images were obtained. With the pre-treatments analyzed, it appeared impossible to find an acceptable balance between ISH signals and preservation of ultrastructural morphology: when good signal-to-noise ratios are obtained, the ultrastructural morphology is already deteriorated. We discuss the parameters that influence the fragile balance between high RNA detection efficiency and good preservation of ultrastructure and the benefit of RCM monitoring in the development and procedures for pre-embedding electron microscopic ISH.

Animals↗

Deposition of nanoparticles in the arterial vessel by porous balloon catheters: localization by confocal laser scanning microscopy and transmission electron microscopy.

Restenosis remains the major limitation of percutaneous transluminal angioplasty (PTA) and stenting in the treatment of patients with atherosclerotic disease. Catheter-based local delivery of pharmacologic agents offers a potential therapeutic approach to reducing restenosis and minimizing undesirable systemic side effects. However, the intramural retention of liquid agents is low. Therefore, to achieve a sustained and regional release of the therapeutic agent it must be encapsulated in nanoparticle carrier systems. The purpose of this study was to investigate the size dependence of the penetration of nanoparticles after local delivery into the vessel wall of the aorta abdominalis of New Zealand white rabbits. Two milliliters of a 0.025% fluorescence-labeled polystyrene nanoparticle suspension with diameters ranging from 110 to 514 nm were infused at 2 atm and at constant PTA pressure of 8 atm into the aorta abdominalis. After the infused segments were removed, the location of nanoparticles was visualized using confocal laser scanning microscopy and transmission electron microscopy. The study demonstrates a size-dependent nanoparticle penetration into the intact vessel wall. While nanoparticles of about 100 and 200 nm were deposited in the inner regions of the vessel wall, 514-nm nanoparticles accumulated primarily at the luminal surface of the aorta. The observations confirm that size plays a critical role in the distribution of particles in the arterial vessel wall. It is additionally influenced by the formation of pressure-induced infusion channels, as well as by the existence of anatomic barriers, such as plaques, at the luminal surface of the aorta or the connective elastic tissue.

Animals↗

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↗

Total internal reflection microscopy and atomic force microscopy (TIRFM-AFM) to study stress transduction mechanisms in endothelial cells.

The cytoskeleton plays a key role in providing strength and structure to the cell. A force balance exists between the cytoskeleton and the extracellular matrix/substratum via the focal contact regions. The purpose of this study is to integrate atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRFM) data to determine the effect of localized force application over the cell surface on the cell's focal contacts size and position. TIRFM gives detailed information on the cell-substrate contact regions and AFM is a tool for elasticity measurements, force application, and topographic surface mapping of the cell. TIRFM data were calibrated by varying the intensity of the evanescent wave to change the interfacial angle at the glass-cell interface. The individual focal contact intensity was found to decrease with increasing interfacial angles from 66 degrees to 80 degrees as the depth of penetration varied from 150 to 66 nm. A measure of cellular mechanical properties was obtained by collecting a set of force curves over the entire cell using the Bioscope AFM. The nuclear region appears to be stiffer than the cell body. Preliminary results of the nanonewtons force application to the cell surface indicate that the cell-substrate contacts rearrange to offset the force. It is evident that the stress applied to the surface is transmitted to the cell-substrate contact region.

Adaptation, Physiological↗

Hürthle-cell lesions of the thyroid: a combined study using transmission electron microscopy, scanning electron microscopy, and immunocytochemistry.

Hürthle cell transformation found in 2 nodular goiters, 2 cases of Hashimoto's thyroiditis, 4 follicular adenomas, 3 follicular carcinomas, 2 papillary carcinomas and 1 anaplastic carcinoma were studied by transmission electron microscopy, scanning electron microscopy and immunocytochemistry. Ultrastructural features of Hürthle cells were identical in non-neoplastic and neoplastic lesions. Cells crammed with mitochondria, showing abnormalities in size, shape and content were prominent in most cases. The presence of distinct smooth-surfaced cells interspersed with cells with many microvilli is almost a pathognomonic scanning electron microscopic feature of benign and malignant Hürthle cell lesions. Most Hürthle cells stained positively for thyroglobulin in all cases, but no immunoreactivity for CEA and calcitonin was found.

Adenocarcinoma↗

Transmission electron microscopy of tissue prepared for scanning electron microscopy by ethanol-cryofracturing.

Tissue processed for scanning electron microscopy by ethanol-cryofracturing combined with critical point drying was embedded and sectioned for transmission electron microscopy. Study of specimens cut in a plane passing through the fracture edge indicated that preservation of cellular fine structure of fractured cells was excellent. Even at the most peripheral edge of the fracture there was no evidence that movement of cytoplasmic components occurred to distort the original structural organization of fractured cells. Lack of cytoplasmic detail in ethanol-cryofractographs has been due more to the nature of the fracturing of the tissue and to the obscuring effects of the metal coating than to structural deformation at the fracture edge or to limitations in resolving power of the scanning electron microscope used.

Animals↗

Imaging and non-contact profile analysis of Nd:YAG laser-irradiated teeth by scanning electron microscopy and confocal laser scanning microscopy.

Two types of non-contact optical profilometers, one performing scanning electron microscopy (SEM) installed with a 3D analyzer and another performing confocal laser scanning microscopy (CLSM) were applied to evaluate imaging and surface profiles simultaneously on enamel and dentin after Nd:YAG laser irradiation. The results were correlated with a stylus profilometer. Surface roughness (Ra) was also measured. Laser was applied perpendicularly to vertical sections of human extracted caries-free molars. Analysis was done on the same spot of the same specimen by both SEM and CLSM for comparison. After irradiation, enamel produced a flake-like surface and dentin resulted in a melted globule surface. SEM and CLSM gave similar surface profiles and different image contrast. The Ra obtained by CLSM was larger than that by SEM. Both SEM and CLSM provided a non-contact evaluation of tooth structural change by laser irradiation through surface analysis in selected micro areas, which was not possible using the stylus profilometer.

Dental Enamel↗

Characterization of exposure to molds and actinomycetes in agricultural dusts by scanning electron microscopy, fluorescence microscopy and the culture method.

Air samples from 79 farms with 10(5) to 10(11) microorganisms/m3 were analyzed by scanning electron microscopy (SEM), fluorescence microscopy (FM), and the culture method. The total exposure to microorganisms (particularly actinomycetes) was underestimated when assessed as colony-forming units (cfu). The average cfu count was one-sixth of the total count according to SEM or FM, and the individual variability was great. This occurrence was partly explained by the aggregation of spores. Single spores accounted for 2-65% of all spores in 35 samples. There was an average of three spores/particle, and 93 (range 67-100)% of the spores were single or in aggregates of respirable size. Aggregation was more pronounced for actinomycetes and at high spore counts. Actinomycetes and bacteria could not be distinguished by FM. Bacteria (other than actinomycetes) were not detected by SEM, yet the total count of microorganisms was similar for FM and SEM. Most particles were spores from actinomycetes and fungi of the genera Aspergillus or Penicillium.

Actinomycetales↗

Different patterns of collagen-proteoglycan interaction: a scanning electron microscopy and atomic force microscopy study.

The extracellular matrix of unfixed, unstained rat corneal stroma, visualized with high-resolution scanning electron microscopy and atomic force microscopy after minimal preliminary treatment, appears composed of straight, parallel, uniform collagen fibrils regularly spaced by a three-dimensional, irregular network of thin, delicate proteoglycan filaments. Rat tail tendon, observed under identical conditions, appears instead made of heterogeneous, closely packed fibrils interwoven with orthogonal proteoglycan filaments. Pre-treatment with cupromeronic blue just thickens the filaments without affecting their spatial layout. Digestion with chondroitinase ABC rids the tendon matrix of all its interconnecting filaments while the corneal stroma architecture remains virtually unaffected, its fibrils always being separated by an evident interfibrillar spacing which is never observed in tendon. Our observations indicate that matrix proteoglycans are responsible for both the highly regular interfibrillar spacing which is distinctive of corneal stroma, and the strong interfibrillar binding observed in tendon. These opposite interaction patterns appear to be distinctive of different proteoglycan species. The molecular details of proteoglycan interactions are still incompletely understood and are the subject of ongoing research.

Animals↗

[Identification of chlamydia and (or) chlamydia-like microorganisms by light microscopy confirmed by electron microscopy and fluorescent in situ hybridization].

Using light microscopy, we have shown that chlamydia and/or chlamydia-like microorganisms are registered in 20-25% of the healthy part of human population, whereas in patients of the same age with gynecological problems these were found in 40-50%. Commonly, the infection was slightly manifested (less than 5% of cells are infected). These results were confirmed in four months but only in heavily infected patients. The light microscope data are confirmed by observations with electron microscopy, and by FISH hybridization of the total chlamydial DNA on cytological preparations with chlamydial inclusions. In some cases, microcolonies revealed by FISH hybridization occupied the majority of the cytoplasm volume. Occasionally, the DNA material was found on the nuclear surface. It seems likely that in heavily infected cells chlamydia are able to penetrate into the perinucular space.

Chlamydia↗

[Guided tissue regeneration: an ultrastructural observation by transmission electron microscopy and scanning electron microscopy].

PTFE membranes are used for guided tissue regeneration in order to treat angular bone defects or forcation involvements in surgical treatment. Ultrastructural investigations have been performed by means of electron transmission and scanning microscopy. In agreement with previous reports, fibroblast cells adhering to the reticular structure of PTFE membrane were observed; these were interposed among coagulated clusters of fibrinous material and blood cells round shaped. Elongated bacterial cells were always present in the microscope fields analysed. These observations were confirmed by means of transmission microscopy; moreover specific techniques enabled us to demonstrate that fibroblast cells were synthetizing collagen, which was present in the form of extracellular fibers mixed to fibrin clusters. Roundish and elongate bacterial cells were always observed both in the extracellular matrix and into macrophages.

Guided Tissue Regeneration, Periodontal↗

Dynamics of epidermal growth factor receptor internalization studied by Nanovid light microscopy and electron microscopy in combination with immunogold labeling.

Individual gold particles with a diameter of approximately 10 to 40 nm can be visualized using video-enhanced contrast microscopy (Nanovid) (De Brabander et al., Cell Motil. Cytoskel. 6, 105-113 (1986)). This technique allows a study of the dynamic properties of receptors and ligands in living cells at high resolution. We have studied epidermal growth factor (EGF) receptor internalization in human epidermoid carcinoma A431 cells, using a monoclonal anti-EGF-receptor antibody conjugated to 20-nm gold particles, referred to as 2E9-gold. Exposure of A431 cells to 2E9-gold at 37 degrees C resulted in binding of the complex at the cell surface. Most of the gold particles exhibit a Brownian type of movement, while a minority appeared immobile. Binding of the 2E9-gold complex is followed by internalization, as judged from Nanovid light microscopy studies in combination with electron microscopic observations. The internalized gold particles clearly cluster into large aggregates, most likely multivesicular bodies. Individual gold particles as well as aggregates are characterized by a saltatory movement, by which the gold particles eventually move from the cell periphery towards the cell center. Addition of EGF results in an increased rate of internalization of 2E9-gold, while Na-azide and nocodazole completely immobilize the intracellular gold particles, as has been demonstrated previously for the transferrin receptor.

Adenosine Triphosphate↗

Endothelialization of a new Dacron graft in an experimental model: light microscopy, electron microscopy and immunocytochemistry.

Two types of synthetic vascular grafts, Dacron Triaxial and Dacron Gelseal Triaxial, were implanted into both the common carotids of sheep. The animals were sacrificed 1, 2, 8, and 16 weeks after surgery. Multiple specimens, obtained from grafts and anastomoses, were studied by light microscopy, transmission and scanning electron microscopy. A parallel immunocytochemical analysis was performed on some specimens. Dacron Triaxial grafts failed to develop a complete neointimal coverage. Myofibroblasts and fibroblasts were the dominant cells in such synthetic graft. Moreover, focal areas of stripping, platelet deposition, and thrombosis were observed at 8 and 16 weeks. In contrast, a stable endothelial coverage developed on the Gelseal Triaxial grafts after 16 weeks.

Animals↗

Combined intravital microscopy and electron microscopy of the blind beginnings of the mesenteric lymphatic capillaries of the rat mesentery. A preliminary report.

The blind beginnings (so-called terminal lymphatics) of the mesenteric lymphatic capillaries have been studied in the rat mesentery by means of intravital microscopy and electron microscopy. Intraluminal objects were observed to move slowly downstream, probably carried by the movement of the lymphatic fluid. No simultaneous changes in capillary lumen could be observed, and the subsequent electron microscope analysis of the same lymphatic capillary demonstrated the absence of smooth muscle cells in the wall of the lymphatic vessel. Interendothelial gaps were present, allowing the uptake of interstitial fluid into the blind ending of the lymphatic capillary.

Animals↗

The collagenous architecture of articular cartilage. Correlation of scanning electron microscopy and polarized light microscopy observations.

The localization and directional orientation of collagen fibers in articular cartilage is demonstrated by scanning electron microscopy and polarized light microscopy. Vertical sections of articular cartilage show different directional orientations of collagen fibers through all zones of cartilage depending upon whether the sections are parallel or perpendicular to the cleft pattern produced when the surface of articular cartilage is pierced with a round pointed awl. Sections parallel to the cleft axis show a significant population of oblique collagen fibers which are not seen in sections perpendicular to the clefts. These oblique fiber groups show a progression from nearly radial to nearly tangential orientation from deep to more superficial zones, with the most abrupt directional change seen through the transitional zone. Within the transitional zone there is a narrow band having no vertical or horizontal collagen fibers and in which collagen fibers intersect predominantly at angles ranging between 45 and 135 degrees. The number of chondrocyte lacunae per unit area is greater in sections parallel to the cleft axis compared to perpendicular sections by a factor of approximately 1.6:1. There is therefore a greater relative number of chondrocytes in the plane of section having the greatest abundance of oblique collagen fibers, suggesting a cellular basis for the collagenous architecture observed. The results are consistent with published biophysical data relating tensile and swelling properties of all zones of articular cartilage to the cleft axis.

Animals↗

The significance of histologic analysis of skin lesions in porphyria cutanea tarda. Light microscopy, electron microscopy, immunohistochemical and immunofluorescence analysis.

Porphyria cutanea tarda (PCT) is one of several entities in the differential diagnosis of scleroderma. We report a 62-year-old man with PCT diagnosed since two decades. Clinical data and the results of light microscopy, immunohistochemistry, immunofluorescence and electron microscopy of skin biopsies are presented. The biopsy revealed sclerosis of the dermis mainly due to increased collagens I and III, and accumulation of collagen IV, which had caused the vessel wall to thicken. Immunofluorescence for detection of immune reactants was negative. It was concluded that the histomorphology of PCT of long duration may be similar to that of scleroderma. Nevertheless, in addition to clinical findings, detailed skin biopsy studies including contemporary techniques can contribute to the differentiation of these diseases.

Adult↗

Vascular interrelationships of near-term mink placenta: light microscopy combined with scanning electron microscopy of corrosion casts.

The microvasculature of the near-term zonary placenta of the mink has been studied using light microscopy and scanning electron microscopy of corrosion casts, prepared from maternal and fetal vessel systems, respectively. The zonary area, most important for placental exchange, includes a characteristic labyrinth. The labyrinth is composed of lobules oriented in a maternal-fetal direction. One maternal stem artery serves as the central axis of a lobule, and about six pairs of fetal stem arteries and stem veins of the chorionic primary villi mark the periphery of the lobule. Viewed from the fetal side of the labyrinth, this lobular structure presents a roughly hexagonal pattern, with the central maternal stem artery and radially oriented arteriolar branches giving the lobule the shape of a star. These arterioles frequently form bridges to neighboring lobular systems; however, the majority continue into the feto-maternally oriented three-dimensional network of maternal capillary sinusoids, which converge on the outlets of the maternal stem veins on the maternal side of the labyrinth. Maternal main crypts are delimited by the rays of the star-shaped lobules containing chorionic primary villi. The latter penetrate into maternal crypts from the fetal side, and are characterized by their axial arterial and venous stem vessels. Fetal secondary villi are arranged at different levels from these stem vessels. The secondary villi are characterized by arterioles and venules branching in pairs from the stem vessels and supply the tributary capillary complexes of terminal villi. The lobular structure of the placental labyrinth provides a three-dimensional framework of vessels where maternal capillary sinusoids and fetal capillaries meet in a one-way cross-current arrangement. The blood flow conditions and the peculiarities of the mink placenta interhemal membrane are compared to those of other carnivores and discussed with respect to the efficiency of the endotheliochorial placenta.

Animals↗