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[Proprioception in the anterior cruciate ligament of the human knee joint--morphological bases. A light, scanning and transmission electron microscopy study].

In 21 human anterior cruciate ligaments removed with their synovial sheaths during autopsy and operation, nerves and nerve endings were demonstrated by light microscopy, scanning electron microscopy and transmission electron microscopy. Ultrastructural examination allowed a classification of nerve endings into three types: Ruffinian corpuscles, Pacinian corpuscles and free (afferent and efferent) nerve endings. The nerve endings corresponded to those characteristic of articular capsules. The anterior cruciate ligament shows evidence of a proprioceptive function besides its stabilizing function, which is based on its collagenous structure. Together, they probably represent the most important factor in the dynamic stabilization of the knee joint. At present it is only possible to speculate on the potential clinical significance of these findings, for example in development of rehabilitation programs and prevention strategies offering protection against anterior cruciate tears. This also applies to the question of whether to preserve the ligament during surgery for ruptures and knee-joint prostheses.

Adult↗

HALE STAIN FOR SIALIC ACID-CONTAINING MUCINS. ADAPTATION TO ELECTRON MICROSCOPY.

The feasibility of using the Hale stain to identify cellular sialic acid-containing mucins by electron microscopy was investigated. Three kinds of mouse ascites tumor cells were fixed in neutral buffered formalin, exposed to fresh colloidal ferric oxide, treated with potassium ferrocyanide, imbedded in Selectron, and sectioned for electron microscopy. Additional staining with uranyl acetate and potassium permanganate was done after sectioning in order to increase contrast. Those cells known to be coated with sialomucin showed deposits of electron-opaque ferric ferrocyanide crystals in the areas where sialomucin concentrations were expected. When these cells were treated with neuraminidase beforehand, these deposits did not appear. It was concluded that, with the precautions and modifications described, the Hale stain can be successfully combined with electron microscopy to identify sialomucin.

Animals↗

The glomerulus in experimental renal disease in rats as observed by light and electron microscopy.

Nephrotoxic serum disease in rats has been studied by light and electron microscopy from 1 hour to 10 weeks after production of the disease. By light microscopy leucocytic infiltration of the glomerular capillary was observed between the 3rd and 6th hour. At 6 hours an increase in colloidal iron-positive material was observed coating the extraluminal surface of the capillaries. Also at this time swelling of the endothelial cells becomes prominent. By 72 hours, thickening of the basement membrane was observed. Glomerular capillary thrombi were observed in approximately half the tissue examined in the first 2 weeks of disease. 50 per cent of the animals showed severe chronic lesions, exudation into the capsular space, crescent formation, and obliteration of glomeruli. At 1 hour electron microscopic pictures showed that osmophilic material may line the foot processes of the epithelial cells and obliterate all but narrow channels of the space between the feet. By 6 hours thickening of the basement membrane was prominent. This change persisted throughout 10 weeks of observation. The tissue from animals which had severe chronic alterations by light microscopy revealed changes which could not be interpreted at this time.

Animals↗

A technique to prepare asbestos air samples for light and electron microscopy.

The direct transfer technique used in preparing waterborne asbestos for analysis by transmission electron microscopy is also suitable for preparing airborne asbestos collected on polycarbonate filters for light microscopy, scanning, and transmission electron microscopy. The same area and even the same fibre may be examined by the three microscopic methods and individual fibres identified by optical and electron-optical techniques and by x-ray microanalysis.

Asbestos↗

Incremental lines in root cementum of human teeth: an approach to their ultrastructural nature by microscopy.

In ground sections of human teeth, root cementum shows under the light microscope as alternating, almost concentric, dark and light rings. In paleontology and forensic medicine, the number of these incremental lines or annulations is used to derive the age-at-death of the individual. To find the ultrastructural features underlying these cemental annulations, we used bright-field light microscopy (LM), confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and electron-dispersive x-radiation (EDX) in a scanning electron microscope (SEM). Annulations visible in ground sections of about 100-micron thickness were no longer visible in semi-thin sections (thickness, 1-2 microns) of the same specimen in the same area. The assumption that annulations could be caused by super-imposing structures in the depth of field of the light microscope's objective lens was not verified by CLSM. Fiber bundles of higher density than the surrounding matrix in TEM micrographs could not be connected unambiguously with annulations in LM micrographs. After all, the ultrastructural nature of cemental annulations remains an open question.

Age Determination by Teeth↗

Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy.

BACKGROUND: The development of multiphoton laser scanning microscopy has greatly facilitated the imaging of living tissues. However, the use of genetically encoded fluorescent proteins to distinguish different cell types in living animals has not been described at single cell resolution using multiphoton microscopy. RESULTS: Here we describe a method for the simultaneous imaging, by multiphoton microscopy, of Green Fluorescent Protein, Cyan Fluorescent Protein and collagen in vivo in living tumors. This novel method enables: 1) the simultaneous visualization of overall cell shape and sub-cellular structures such as the plasma membrane or proteins of interest in cells inside living animals, 2) direct comparison of the behavior of single cells from different cell lines in the same microenvironment in vivo. CONCLUSION: Using this multi-fluor, multiphoton technique, we demonstrate that motility and metastatic differences between carcinoma cells of differing metastatic potential can be imaged in the same animal simultaneously at sub-cellular resolution.

Animals↗

Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy.

Although the addition of just the excitation light field at the focus, or of just the fluorescence field at the detector is sufficient for a three- to fivefold resolution increase in 4Pi-fluorescence microscopy, substantial improvements of its optical properties are achieved by exploiting both effects simultaneously. They encompass not only an additional expansion of the optical bandwidth, but also an amplified transfer of the newly gained spatial frequencies to the image. Here we report on the realization and the imaging properties of this 4Pi microscopy mode of type C that also is the far-field microscope with the hitherto largest aperture. We show that in conjunction with two-photon excitation, the resulting optical transfer function displays a sevenfold improvement of axial three-dimensional resolution over confocal microscopy in aqueous samples, and more importantly, a marked transfer of all frequencies within its inner region of support. The latter is present also without the confocal pinhole. Thus, linear image deconvolution is possible both for confocalized and nonconfocalized live-cell 4Pi imaging. Realized in a state-of-the-art scanning microscope, this approach enables robust three-dimensional imaging of fixed and live cells at approximately 80 nm axial resolution.

Equipment Design↗

Variable incidence angle fluorescence interference contrast microscopy for z-imaging single objects.

Surface-generated structured illumination microscopies interrogate the position of fluorescently labeled objects near surfaces with nanometer resolution along the z axis. However, these techniques are either experimentally cumbersome or applicable to a limited set of experimental systems. We present a new type of surface-generated structured illumination fluorescence microscopy, variable incidence angle fluorescence interference contrast microscopy (VIA-FLIC), in which the fluorescent sample is assembled above a reflective Si surface and the incidence angle of excitation light is varied by placing annular photomasks with different radii in the aperture diaphragm plane of the microscope. The variation in incidence angle alters the interference pattern of excitation light, and hence the intensity of detected fluorescence. Quantitative VIA-FLIC is tested by using a set of fluorophore-containing supported membranes separated from the Si surface by SiO2 layers of variable thicknesses. The resulting fluorescence intensity versus incidence angle curves depends on the separation from the Si surface and when fit with an appropriate model yield precise SiO2 thicknesses that are accurate with respect to the known SiO2 thicknesses. Since only a simple modification to a standard epifluorescence microscope is required, VIA-FLIC offers a versatile method to produce z-reconstructions with high resolution for a wide range of biological systems.

Algorithms↗

Applications of combined spectral lifetime microscopy for biology.

Live cell imaging has been greatly advanced by the recent development of new fluorescence microscopy-based methods such as multiphoton laser-scanning microscopy, which can noninvasively image deep into live specimens and generate images of extrinsic and intrinsic signals. Of recent interest has been the development of techniques that can harness properties of fluorescence, other than intensity, such as the emission spectrum and excited state lifetime of a fluorophore. Spectra can be used to discriminate between fluorophores, and lifetime can be used to report on the microenvironment of fluorophores. We describe a novel technique-combined spectral and lifetime imaging-which combines the benefits of multiphoton microscopy, spectral discrimination, and lifetime analysis and allows for the simultaneous collection of all three dimensions of data along with spatial and temporal information.

Animals↗

A method for the examination of the same cell using light, scanning and transmission electron microscopy.

A method is described for preparing the same cell from a cytospin preparation for comparative investigation by light microscopy, scanning electron microscopy and transmission electron microscopy. A permanent numbered grid pattern was etched on a glass microscope slide to facilitate cell location in each microscopic mode. Data from one cell or group of cells was thus obtained from three sources. This method provides a useful adjunct to routine cytological diagnosis.

Cells↗

Fluorescent photoreceptors of transgenic Xenopus laevis imaged in vivo by two microscopy techniques.

PURPOSE: To develop a method for imaging individual photoreceptors in an intact transgenic Xenopus eye, thus allowing in vivo observation of the effects of various transgenes on photoreceptor development, degeneration, or both. METHODS: Albino and pigmented transgenic Xenopus laevis that express enhanced green fluorescent protein (GFP) in the major ("red") rods were generated. The distribution of GFP throughout the retina and within the rods was evaluated by confocal microscopy of frozen sections and immunoelectron microscopy. In vivo images of photoreceptors were obtained using conventional fluorescence microscopes to image through the lens of the eye or a laser scanning confocal microscope to image through the hypopigmented iris of albino eyes. RESULTS: Confocal and immunoelectron microscopy of tissue sections showed that GFP was predominantly localized to the inner segments of the major rods; a smaller amount was in the outer segments. In a number of animals, not all the major rods expressed GFP. It was possible to identify these animals by obtaining fluorescence images of the retinas of intact, living tadpoles with conventional fluorescence microscopes, using the lens of the tadpole as part of the optical path. Confocal images of living animals could be used to visualize the distribution of GFP within the photoreceptors. CONCLUSIONS: The ability to observe individual photoreceptors noninvasively allows in vivo longitudinal microscopic analysis of photoreceptor development in transgenic Xenopus tadpoles.

Animals↗

Fluorescence microscopy for the evaluation of the margins of Class V restorations in vitro.

PURPOSE: First, to elucidate the cause of the appearance of "white lines" in Class V restorations as seen under the light microscope and their enhancement when using a fluorescent microscope (FM); second, to compare the results of the FM quantitative marginal analysis to those of the SEM quantitative marginal analysis. MATERIALS AND METHODS: Thirty-two standardized Class V fillings, with half of the preparation in dentin and half in enamel, were placed in 16 human caries-free premolars on the buccal and lingual surface using 4 adhesive systems (Clearfil SE, Syntac, 2 experimental self-etching adhesives) and the composite Tetric Ceram. All teeth were connected to a device containing horse serum to simulate dentinal fluid and subjected to thermomechanical loading (1,200,000 cycles at 49 N, 3000 cycles at 5 degrees C/55 degrees C). The restoration margins of 24 fillings were directly evaluated with FM--after the fabrication of replicas--and with SEM. Eight fillings were used to elucidate the cause of the fluorescent signal, by examining them with FM and different filters under dry and moist conditions, and also with dark field microscopy and CLSM. Further, two Class V fillings with the same composite but without the fluorescent substances were made and evaluated. Selected specimens were cut in the bucco-oral direction to analyze the restorative interface on section replicas with SEM. To detect differences between FM and SEM, the Wilcoxon test was performed (p < 0.05). To evaluate the degree of agreement between the two evaluation methods, a linear regression analysis was performed and the Spearman correlation coeffcient was calculated. RESULTS: The evaluation of Class V restorative margins by SEM, dark field microscopy, and CLSM revealed that the phenomenon seems to be an optical effect caused by the debonding of the composite from the underlying substrate creating another refractive environment when air penetrates into the gap. The effect depends on the geometry of the cavity design, the absence of moisture, and the use of a fluorescent composite material. The light source that produced the best visible results covered both ultraviolet and the blue part of white light (350 to 460 nm), producing light green lines and green areas. Those green or white areas as seen with the light microscope were related to gaps in the interface between tooth substance and composite. With the exception of one of the experimental self-etching adhesives, the mean percentage of continuous margin of the 4 groups showed no statistically signifcant dfference for the two test methods. Linear regression analysis revealed a good correlation between the two test methods for dentin margins and a moderate correlation for enamel margins. CONCLUSION: Fluorescence microscopy seems to be a useful tool for evaluating dentinal and enamel margins of Class V restorations in vitro.

Adhesives↗

Optical and scanning electron microscopy in the single osteoclast resorption assay.

The present studies relate to the single or isolated osteoclastic resorption function assay which we introduced in 1983 to overcome objections to assays based upon measurements of calcium release from bones, in which it was never strictly controlled whether the mechanism involved the destruction of bone with the formation of classical Howship's lacunae. The method may prove to be quite popular in the near future and has already been adopted by other research groups. In previous work, we had utilised stereophotogrammetry of scanning electron micrographs to measure the depth, volume and other parameters of the individual lacunae. However, increasing experience with the method has suggested that we can await a wide range of biological variability in single cell function in any one experiment. We have therefore tested other methods from which data could be obtained more rapidly to permit a better statistical analysis, albeit with reduced accuracy, of each resorption complex. The main aim of the studies reported here was to evaluate various methods of optical and scanning electron microscopy that can be used for the visualization of osteoclasts and their associated resorption lacunae generated in vitro in slabs of dentine and bone. Optical microscopy was found to be complementary to SEM, enabling vital microscopy of unstained and stained cells. In particular, oblique illumination LM and tandem scanning reflected LM (TSRLM) proved to be of paramount value for this purpose. Fixed coated specimens could be most rapidly scanned for resorption lacunae using darkfield reflected LM or TSRLM.

Animals↗

[Vital microscopy of the lung].

In order to develop an optimal method for prevital microscopy of the lung, in 12 experiments performed in mongrel dogs advantages of three methods of contact microscopy were compared: dark-field, polarization and fluorescent-absorptive. The results on investigating the lung microstructure and microcirculation demonstrated usefulness of all the three methods. Nevertheless, in order to estimate the lung microstructure and microcirculation, the dark-field method is the most expedient, to reveal the pleural vascular network, the best results are obtained with the fluorescent-absorptive microscopy. It is necessary to fix the part examined under the objective by means of a special fixator-sticker that does not traumatize the lung parenchyma and does not disturb the respiratory microcirculation.

Animals↗

Direct evidence of plastid DNA and mitochondrial DNA in sperm cells in relation to biparental inheritance of organelle DNA in Pelargonium zonale by fluorescence/electron microscopy.

The fate of plastid DNA and mitochondrial DNA was followed during spermatogenesis in thin sections of the pollen grains in Pelargonium zonale by epifluorescence microscopy, after staining with 4'-6-diamidino-2-phenylindole (DAPI), by the combination of fluorescence and electron microscopic images by color image processor, and by immunogold electron microscopy. DAPI-stained thin sections showed that there are two different types of fluorescent spots due to organelle DNA in the cytoplasm of generative cells and sperm cells: spherical spots that emit strong fluorescence (SF spots) and a ring-shaped group of tiny spots that emit a pale fluorescence (RG spots). The RG spots correspond to peanut-shaped refractive dense bodies under a phase-contrast microscope. Combination of the fluorescence and electron microscopic images by a color image processor showed that SF spots are not emitted from peanut-shaped electron-dense bodies but are from small spherical granules with cristae-like lamellae. Immunogold electron microscopy using ultrathin sectioning showed that the small spherical granules are mitochondria with typical cristae and that the peanut-shaped electron-dense bodies are plastids. The results clearly indicate that mitochondria and plastids with DNA molecules are present in the cytoplasm of sperm cells. The presence of these plastid DNA molecules may be responsible for the biparental inheritance of plastids. In addition, mitochondrial DNA may also show biparental inheritance.

DNA, Mitochondrial↗

In vitro destruction of nerve cell cultures by Acanthamoeba spp.: a transmission and scanning electron microscopy study.

Trophozoites of 4 species of Acanthamoeba were cytopathic for cultured rat B103 neuroblastoma cells. Cytopathogenicity was evaluated by a chromium release assay and by transmission and scanning electron microscopy. Acanthamoeba culbertsoni, Acanthamoeba castellanii, and Acanthamoeba polyphaga destroyed B103 target cells at 37 C as evidenced by the release of radiolabel. Acanthamoeba astronyxis did not produce cytopathology at 37 C but destroyed nerve cells at 25 C. Transmission and scanning electron microscopy of cocultures maintained at different time periods revealed that all species of Acanthamoeba exhibited long cylindrical structures, termed digipodia, which made contact with target cells. Following this effector cell-target cell contact, membrane blebbing on the nerve cells was observed. These events were followed either by lysis of target nerve cells or ingestion of the target cells via food-cups and their subsequent channeling into intracytoplasmic food vacuoles. Use of the TUNEL (TdT-mediated dUTP nick end labeling) technique indicated that approximately 40% of B103 cells incubated with A. culbertsoni, 20% of B103 cells cocultured with A. castellanii or with A. polyphaga, and less than 1% of B103 cells incubated with A. astronyxis at 37 C were apoptotic after 24 hr of coculture. Studies using electron microscopy indicated that Acanthamoeba trophozoites destroyed nerve cells both by cytolysis and by ingestion of whole nerve cells via food-cups.

Acanthamoeba↗

Comparative scanning, transmission and atomic force microscopy of the microtubular cytoskeleton in fenestrated liver endothelial cells.

Endothelial fenestrae control the exchange of fluids, solutes and particles between the sinusoidal lumen and the microvillous surface of the parenchymal cells. Fenestrae have a critical dimension in the order of 150-200 nm, making it necessary to use microscopes with a resolution better than the light microscope. Comparative whole-mount preparations of isolated, purified and cultured rat liver sinusoidal endothelial cells (LEC) were studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). Examination of detergent-extracted LEC by SEM and TEM shows an integral cytoskeleton: sieve plates are delineated by a sieve plate-associated cytoskeleton ring and fenestrae by a fenestrae-associated cytoskeleton ring. By using microtubule altering agents we could demonstrate: (1) the architectural role of microtubules in arranging fenestrae, (2) the existence of a population of microtubules resistant against low temperature and colchicine, (3) the ability of LEC to shift the microtubule assembly-disassembly steady state under various conditions, (4) and the necessity of an intact microtubular cytoskeleton to support the increase in the number of fenestrae after cytochalasin B. Topographical examinations of AFM images revealed that sieve plates are delineated by elevated borders, probably projections of the underlying tubular cytoskeleton.

Animals↗

Correlated confocal and intermediate voltage electron microscopy imaging of the same cells using sequential fluorescence labeling, fixation, and critical point dehydration.

Confocal laser scanning microscopy (CLSM) and intermediate voltage transmission electron microscopy (IVEM) each has its own particular advantages. CLSM can examine living cells, but is particularly useful when applied to cells that have been lightly fixed, permeabilized, and stained with fluorescent-labeled antibodies for localization of specific molecular species at the resolution of the light microscope while still in the hydrated state. IVEM provides much higher resolution images, but requires more drastic preparation procedures, including dehydration. This paper presents methods for combining these complementary approaches to examine exactly the same cells sequentially by CLSM and IVEM. Cells are grown in culture on sterile formvar films spread over gold index grids on cover glasses, which are mounted on larger cover glasses or microscope slides with spacers to prevent compression of the cells. Light and epifluorescence microscopy, and CLSM are performed concentrating on cells in grid openings. Then the grids are fixed with aldehydes followed by OsO4, dehydrated and critical point dried (CPD) from liquid CO2. Immediately following CPD, the grids are ready for examination in the IVEM. Low magnification (300-600x) survey images allow correlation of the IVEM images with the light microscopic images. In higher power images, structures that are fluorescent labeled can be related to corresponding regions in the IVEM images.

Animals↗