Scanning microscopy最新文献

Regeneration of the synovial lining in the synovectomized rabbit knee was studied using the scanning electron microscope. The resected synovia regenerated considerably 3 weeks after synovectomy. However, 44 weeks following excision, their surface morphology was still very different from that of the normal tissue of intact animals. The regenerated synovia were characterized by three main features: the large number of various patterns, the many fields harboring fibrillation and the almost total lack of a bubble layer (the latter was formerly shown to be predominant on normal, intact synovia). The surface morphology of the non-operated (contralateral) knee differed greatly from that of normal synovia. The surface of sham-operated synovia was totally covered by the bubble layer. The appearance of vast fields harboring fibrillations indicated deficient ultrastructural regeneration. The altered surface morphology of the contralateral synovia was a novel finding. We wonder whether it would be appropriate to propose that the systemic reaction induced by synovectomy of the experimental knee initiated the synovial appearances recorded on the contralateral knee. The data reported here rule out the possibility of using the contralateral leg as an intact control.

Backscattered electron (BSE) imaging was used to display heavy metal stained biological structures of various embedded specimens. Samples were fixed, stained and embedded in resin blocks as with preparation for the transmission electron microscope (TEM). Blocks were trimmed to center the specimens in a trapezoidal face of up to 5 mm2 and their sides painted with conductive silver paint leaving the face uncovered. Blocks were sputter coated with 6-8 nm of silver, chromium or aluminum, with aluminum providing the best specimen contrast in BSE mode. Samples were examined in a field emission scanning electron microscope operated at a high emission current of 50 microA. Both the fixation protocol and microscope operating parameters were optimized to maximize the number of BSE available from the smallest probe. An accelerating voltage of 10 keV was found optimal for resolution and contrast. The technique allowed the direct visualization of embedded samples at resolutions beyond light microscopy with good contrast, without cutting sections, and avoiding grid bars obscuring areas of interest. The two dimensional images provided averaged information on the internal structures of the specimens in relation to the predicted emission depth of the BSE. The technique could be used for rapid diagnostics in pathological examinations, or for routine preselection of areas of interest within a sample face before final trimming for ultrathin sectioning for higher resolution TEM study.

The present investigation was designed to study interactions between Madin-Darby canine kidney (MDCK) cells and calcium oxalate monohydrate (COM) crystals and to clarify the significance of these crystal-cell interactions in stone pathogenesis. MDCK cells cultured in the presence of COM crystals showed a time-dependent uptake of crystals; this was specific for COM crystals. In the dynamic model system designed to study these phenomena under more physiological conditions, COM crystals adhered to the cell surface and were subsequently internalized. In this endocytotic process, the microvilli of the cell appeared to play an important role. The observation by scanning electron microscopy of complexes consisting of aggregated COM crystals and cell debris led us to speculate that adhesion and endocytosis of crystals might provide the calculus nidus for aggregation and retention of crystals in the renal tubule. Furthermore, glycosaminoglycans and the macromolecular fraction of human urine were shown to have the ability to inhibit the cellular uptake of crystals. Evidence that similar processes may also occur in vivo was obtained using an experimental stone model in rats. Our experiments revealed that most of the COM crystals adhered to the tubular cells and some crystals were endocytosed by the cell. Thus, these crystal-cell interactions might be one of the earliest processes in the formation of kidney stones. Further elucidation of the mechanism and the regulatory factors involved in this process may provide new insight into stone pathogenesis.

Seventy-one chick embryos of both sexes at the 35 Hamburger and Hamilton (H-H) developmental stage were processed for scanning electron microscopy of vascular corrosion casts and of critical point dried specimens, as well as transmission electron- and light microscopy, in order to study the angiogenic structures. The gonadal subepithelial capillary network was located at the level of the tunica albuginea under the covering epithelium. The casts showed a densely-meshed capillary network and numerous sprouting (nodular protrusions or capillary sprouts) and non-sprouting (enlarged vessels and angiogenic holes) angiogenic structures that were randomly distributed and mixed. Four types of angiogenic holes were encountered in the casts: primary (diameter < 2.5 microns), secondary (diameter > 2.5 microns), tertiary (variable diameter and circular narrowings on one side), and open angiogenic holes. We suggest that the different morphologies reflect evolution of these holes. Furthermore, the open angiogenic hole would probably either form nodular protrusions at its open ends, which tend to join with other nodular protrusions and neighboring capillaries and form new vessels; or there would be fusion with two or more neighboring open holes. Correlative critical point dried sections showed fenestrations in the capillary walls and transcapillary pillars that corresponded to the angiogenic holes found in the casts. Ultrathin sections of the vessels presented typical characteristics of growing endothelium: large nuclei with loosely textured chromatin, abundant cytoplasm rich in cell organelles and intraluminal endothelial processes.

We have developed scanning near-field optical/atomic force microscopy (SNOM/AFM). The SNOM/AFM uses a bent optical fiber simultaneously as a dynamic force AFM cantilever and a SNOM probe. Resonant frequency of the optical fiber cantilever is 15-40 kHz. Optical resolution of the SNOM/AFM images shows less than 50 nm. The SNOM/AFM system contains photon counting system and polychrometer/intensified coupled charge devise (ICCD) system to observe fluorescence image and spectrograph of micro areas, respectively. Cultured cells were stained with fluorescein isothiocyanate (FITC)-labeled anti-keratin antibody or FITC-labeled phalloidin after treatment with Triton X-100. Fluorescence and topographic images were obtained in air and water. The fluorescence images showed clear images of keratin and actin filaments. The SNOM/AFM is perfect to observe biomaterials in liquid with a liquid chamber while the topographic Images showed subcellular structures which correspond to keratin and actin filaments.

Scanning electron microscopy (SEM) of red blood cells in whole blood samples from rats was performed following acute gamma-irradiation of animals with 0.25 to 1 Gy. Increased incidence of echinocytosis was observed and found to be dose- and time-dependent. At a higher radiation dose (1 Gy), echinocytosis was revealed within 5 minutes after treatment and persisted up to 3 weeks. The data demonstrate the applicability of SEM for detecting minimal radiation-induced lesions of red blood cells.

To better understand urinary inhibitors of calcium oxalate crystallization, both zeta potential measurement and particle size analysis were chosen to illustrate: (1) the potential therapeutic efficacy of G872, a semi-synthetic sulfated polysaccharide, in stone prevention; and (2) the relative contribution of various urinary fractions ¿e.g., ultrafiltered urine (UFU), Tamm-Horsfall protein (THP), urinary polyanions precipitated with cetylpyridinium chloride (CPC), urinary macromolecular substances with different concentration ratios (UMS10,50,90 and UMS'10,50,90) and THP-free urine (THPFU)¿ to total urinary inhibitory activity. The results showed: (1) addition of G872 significantly enhances urinary inhibitory activity and negative zeta potential values; (2) re-addition of the CPC to UFU completely restores urinary inhibitory activity; and (3) artificial urines prepared by mixing UMS'10,50,90 from THPFU with UFU differed in inhibitory activity from that prepared by mixing UMS10,50,90 from a pooled normal urine with UFU. Based on these experimental results, the following speculations can be made: (1) normal human urines are considered to be a protective colloidal system; (2) urinary inhibitory activity originates mainly from CPC and/or UMS; (3) normal THP is a protective material to maintain urinary inhibitory activity; and (4) mutual interaction between urinary inhibitors may change the total urinary inhibitory activity.

Physiological elimination of unwanted cells within the organism occurs via cell death by apoptosis and phagocytosis of these cells represents a key event in the apoptotic process. Macrophages, which are the dedicated phagocytes, and other occasionally phagocytic cells ingest the apoptotic cells while they are still intact, thus preventing the leakage of potentially harmful materials from the dying cells. Although evidence has been presented that the elimination of apoptotic bodies from the tissue operates by means of specific recognition systems, the molecular mechanisms by which an apoptotic cell is recognized are poorly understood. Recent data indicate that phagocyte recognition of apoptotic cells involves at least four classes of receptors on the phagocyte surface. On the other side, dying cells may display different signals to signal their status. Exposure of phosphatidyl serine (PS) on the surface of apoptotic lymphocytes triggers their specific recognition and removal by macrophages. Apoptotic thymocytes are also identified by altered lipid packing on their surface. Different populations of macrophages use either the vitronectin receptor or the PS receptor to recognize and remove apoptotic cells. It has been suggested that the asialoglycoprotein and the galactose-specific receptors of healthy hepatocytes and sinusoidal liver cells are implicated in the engulfment of apoptotic hepatocytes, likely in cooperation with other hepatic carbohydrate-specific receptor systems. The purpose of this review is to examine current knowledge of the mechanisms by which phagocytes recognize and ingest apoptotic cells.