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At least 937 records · Page 52Linked to original sources
PubMed1994

Direct visualization of lipid aggregates in native human bile by light- and cryo-transmission electron-microscopy.

The evolution of microstructures present in human gallbladder and hepatic bile was observed simultaneously by video-enhanced light microscopy (VELM) and transmission electron microscopy of vitrified specimens (cryo-TEM), as a function of time after withdrawal from patients. Fresh centrifuged gallbladder bile samples contained small (6 nm) spherical micelles in coexistence with vesicles (40 nm). Out of the seven bile samples investigated four contained, in addition, two types of elongated aggregates that have not been previously described. Uncentrifuged gallbladder bile also contained a mixture of ribbon- and plate-like crystals seen by VELM, but not by cryo-TEM. In aged (3-6-week-old) gallbladder bile samples VELM also revealed spiral and helical crystal structures. No such crystals were present in hepatic bile samples, although microcrystals, not observable by VELM were seen by cryo-TEM in addition to micelles and vesicles. The similarity of these observations to those observed in bile models lends strong support for the validity of the model systems. Furthermore, the presence of microcrystals in hepatic bile samples, apparently devoid of crystals by light microscopy, indicates that under certain conditions the common criterion of 'nucleation time' (NT), based on light microscopy, does not represent the real time of nucleation. In the human bile samples investigated in this study the dissociation between NT and the time of observation of microcrystals was seen in hepatic but not in gallbladder bile samples. Hence, crystal growth may be rate limiting only in dilute biles.

Bile↗
PubMed1990

Combined conventional transmission, scanning, and high-voltage electron microscopy of the same blood vessel for the study of targeted inflammatory cells in blood-brain barrier inflammation.

The microvasculature of brains and spinal cords from mice subjected to chronic relapsing experimental autoimmune encephalomyelitis (CREAE) was studied using three different electron microscopic techniques. Blood vessels were initially examined by scanning electron microscopy. This allowed for the investigation of topographical changes of the luminal aspects of endothelial cells (ECs) and identification of targeted inflammatory cells (ICs) attached to the ECs. The same blood vessel areas with attached ICs examined by scanning electron microscopy were subsequently trimmed, processed for routine conventional transmission electron microscopy, and plastic embedded. Thin (80 nm) sections were cut and evaluated. Semithick (0.5-0.75 microns) serial sections of this material were examined by high-voltage electron microscopy. Data presented here described a useful technique for combining several ultrastructural techniques that permits simultaneous topographic and cross-sectional examination of selected regions of individual blood vessels or specifically targeted ICs.

Animals↗
PubMed2005

Watching the components of photosynthetic bacterial membranes and their in situ organisation by atomic force microscopy.

The atomic force microscope has developed into a powerful tool in structural biology allowing information to be acquired at submolecular resolution on the protruding structures of membrane proteins. It is now a complementary technique to X-ray crystallography and electron microscopy for structure determination of individual membrane proteins after extraction, purification and reconstitution into lipid bilayers. Moving on from the structures of individual components of biological membranes, atomic force microscopy has recently been demonstrated to be a unique tool to identify in situ the individual components of multi-protein assemblies and to study the supramolecular architecture of these components allowing the efficient performance of a complex biological function. Here, recent atomic force microscopy studies of native membranes of different photosynthetic bacteria with different polypeptide contents are reviewed. Technology, advantages, feasibilities, restrictions and limits of atomic force microscopy for the acquisition of highly resolved images of up to 10 A lateral resolution under native conditions are discussed. From a biological point of view, the new insights contributed by the images are analysed and discussed in the context of the strongly debated organisation of the interconnected network of membrane-associated chlorophyll-protein complexes composing the photosynthetic apparatus in different species of purple bacteria.

Bacteria↗
PubMed2005

Direct observation of frits and dynamic air bubble formation in capillary electrochromatography using confocal fluorescence microscopy.

Confocal fluorescence microscopy has been used to study the capillary electrochromatography (CEC) frits and dynamic air bubble formation under real chromatographic conditions. Confocal fluorescence microscopy provides a nondestructive way to view the three-dimensional structure of the frits with high spatial resolution. Frits prepared with four different procedures were studied: (1) sintering bare silica beads with sodium silicate; (2) sintering bare silica beads wetted with water; (3) sintering C18 beads wetted with water; and (4) sintering C18 beads wetted with water and then surfaced-recovered with C18. Frits prepared with sintering silicate-wetted beads have a high degree of heterogeneity, while the other three types of frits have similar, more homogeneous packing structures. Confocal fluorescence microscopy also provides sufficient temporal resolution for in situ observation of the dynamic processes in air bubble formation. In this study, air bubble formation is imaged during the reorganization process of the packing bed and is shown to occur close to the border between the packing bed and the outlet frit. Confocal fluorescence microscopy opens a new avenue in studying dynamic processes in situ in CEC separations.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2004

Detection and quantitation of human immunodeficiency virus type-1 particles by confocal microscopy.

A method is described to visualise directly human immunodeficiency virus type-1 (HIV-1) particles. HIV-1 containing samples were adsorbed onto a plastic surface and doubly labeled with antibodies specific for viral proteins and sensitive nucleic acids dyes. Laser scanning confocal microscopy detected co-localization of viral proteins and nucleic acids, thus allowing specific identification of HIV. Using this technique, we have quantified eight different HIV-1 sub-types and three HIV-1 groups in tissue culture supernatants from infected peripheral blood mononuclear cells (PBMCs). Confocal counts correlated well with electron microscopy (EM) counts and HIV-1 RNA loads as determined by quantitative PCR. Confocal microscopy may prove to be a simple alternative to electron microscopy for virus identification and quantitation.

Cell Line↗
PubMed2006

Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy.

Acetylcholine receptor (AChR) supramolecular aggregates that have hitherto only been accessible to examination by electron microscopy were imaged with stimulated emission depletion (STED) fluorescence microscopy, providing resolution beyond limits of diffraction of classical wide-field or confocal microscopes. We examined a Chinese hamster ovary cell liner CHO-K1/A5, that stably expresses adult murine AChR. Whereas confocal microscopy displays AChR clusters as diffraction-limited dots of approximately 200 nm diameter, STED microscopy yields nanoclusters with a peak size distribution of approximately 55 nm. Utilizing this resolution, we show that cholesterol depletion by acute (30 min, 37 degrees C) exposure to methyl-beta-cyclodextrin alters the short and long range organization of AChR nanoclusters on the cell surface. In the short range, AChRs form larger nanoclusters, possibly related to the alteration of cholesterol-dependent protein-protein associations. Ripley's K-test on STED images reveals changes in nanocluster distribution on larger scales (0.5-3.5 microm), which possibly are related to the abolition of cytoskeletal physical barriers preventing the lateral diffusion of AChR nanoclusters.

Algorithms↗
PubMed2004

Evaluation of microscopic techniques (epifluorescence microscopy, CLSM, TPE-LSM) as a basis for the quantitative image analysis of activated sludge.

Microscopic techniques ranging from epifluorescence microscopy to confocal laser scanning microscopy (CLSM) and two photon excitation laser scanning microscopy (TPE-LSM) combined with fluorescent stains can help to evaluate complex microbial aggregates such as activated sludge flocs. To determine the application limits of these microscopic techniques, activated sludge samples from three different sources were evaluated after staining with a fluorescent viability indicator (Baclight Bacterial Viability Kit, Molecular Probes). Image analysis routines were developed to quantify overall amounts of red and green stained cells, location of stained cells within the flocs, and the spatial organization in clusters and filaments. It was found that the selection of the appropriate microscopic technique depends strongly on the type of microbial aggregates being analyzed. For flocs with high cell density, the use of TPE-LSM is preferred, since it provides a clearer image of the internal structure of the aggregate. Epifluorescence microscopy did not allow to reliably quantify red stained cells in dense aggregates. CLSM did not adequately image the internal filamentous structure and the location of stained cells within dense flocs. However, for typical activated sludge flocs epifluorescence and CLSM proved adequate.

Flocculation↗
PubMed2005

Confocal microscopy: imaging cervical precancerous lesions.

OBJECTIVES: We explore the clinical potential of reflectance and fluorescence confocal microscopy to image the morphologic and biochemical changes associated with precancer, in order to aid in the detection and diagnosis of cervical dysplasia. METHODS: Cervical epithelial tissue samples imaged ex vivo or in vivo were obtained from M. D. Anderson Cancer Center and Lyndon B. Johnson Hospital in Houston, Texas. Confocal reflectance microscopy was used to image ex vivo cervical biopsies and in vivo cervical tissue. Confocal fluorescence microscopy was used to image ex vivo cervical tissue slices. RESULTS: We present reflectance and fluorescence confocal images of cervical tissue demonstrating the ability to differentiate between normal and abnormal cervical tissue. CONCLUSIONS: We believe that there is significant clinical potential for confocal microscopy to provide a sensitive and specific method for cervical precancer detection.

Biopsy↗
PubMed2001

Three-dimensional imaging of embryonic mouse kidney by two-photon microscopy.

Developing mammalian embryonic kidney becomes progressively more elaborate as the ureteric bud branches into undifferentiated mesenchyme. Morphological perturbations of nephrogenesis, such as those seen in inherited renal diseases or induced in transgenic animals, require careful and often tedious documentation by multiple methodologies. We have applied a relatively quick and simple approach combining two-photon microscopy and advanced three-dimensional (3-D) imaging techniques to visualize and evaluate these complex events. As compared with laser confocal microscopy, two-photon microscopy offers superior optical sectioning deep into biological tissues, permitting analysis of large, heterogeneous, 3-D structures such as developing mouse kidney. Embryonic and newborn mouse kidneys were fluorescently labeled with lectins, phalloidin, or antibody. Three-dimensional image volumes were then collected. The resulting volume data sets were processed using a novel 3-D visualization technique. Reconstructed image volumes demonstrate the dichotomous branching of ureteric bud as it progresses from a simple, symmetrical structure into an elaborate, asymmetrical collecting system of multiple branches. Detailed morphology of in situ cysts was elucidated in a transgene-induced mouse model of polycystic kidney disease. We expect this integration of two-photon microscopy with advanced 3-D image analysis will provide a powerful tool for illuminating a variety of complex developmental processes in multiple dimensions.

Animals↗
PubMed1999

Development of X-ray excitable luminescent probes for scanning X-ray microscopy.

Transmission soft X-ray microscopy is now capable of achieving resolutions that are typically 5 times better than the best-visible light microscopes. With expected improvements in zone plate optics, an additional factor of two may be realized within the next few years. Despite the high resolution now available with X-ray microscopes and the high X-ray contrast provided by biological molecules in the soft X-ray region (lambda = 2-5 nm), molecular probes for localizing specific biological targets have been lacking. To circumvent this problem, X-ray excitable molecular probes are needed that can target unique biological features. In this paper we report our initial results on the development of lanthanide-based fluorescent probes for biological labeling. Using scanning luminescence X-ray microscopy (SLXM, Jacobsen et al., J. Microscopy 172 (1993) 121-129), we show that lanthanide organo-polychelate complexes are sufficiently bright and radiation resistant to be the basis of a new class of X-ray excitable molecular probes capable of providing at least a fivefold improvement in resolution over visible light microscopy. Lanthanide probes, able to bind 80-100 metal ions per molecule, were found to give strong luminescent signals with X-ray doses exceeding 10(8) Gy, and were used to label actin stress fibers and in vitro preparations of polymerized tubulin.

Actins↗
PubMed1997

Histologic analysis of thermal effects of laser thermokeratoplasty and corneal ablation using Sirius-red polarization microscopy.

PURPOSE: To evaluate how well several histologic techniques differentiate degrees of thermally induced changes in corneal tissue after laser thermokeratoplasty (LTK) or corneal ablation. SETTING: Medical Laser Center Lübeck, Germany. METHODS: Corneas of freshly enucleated porcine eyes were treated with a continuous wave laser diode (1.86 microns) and a pulsed chromium-thulium-holmium: YAG laser (2.1 microns) to produce LTK lesions or ablated with a Q-switched and a free-running chromium-erbium: YSGG laser (2.70 microns), a free-running erbium: YAG laser (2.94 microns), and an argon-fluoride excimer laser (193 nm). The lesions were evaluated by light microscopy (LM) (hematoxylin and eosin, Azan, van Gieson's, and Masson-Goldner's trichrome stains), transmission electron microscopy (TEM), and polarization microscopy after Sirius-red staining. Sirius-red, a strongly elongated, birefringent molecule binding parallel to collagen molecules, was used to enhance corneal birefringence. RESULTS: With routine LM, it was difficult to discriminate the degrees of thermal alterations in LTK lesions. Combined Sirius-red staining and polarization microscopy distinguished between a strongly coagulated central zone and the transition zone to normal tissue. Sirius-red uptake was increased in both zones, reflecting the availability of new binding sites. The central zone appeared darker under polarization than normal collagen because of a loss of birefringence. Intrinsic birefringence was greatly reduced; however, form birefringence partly remained as long as some collagen fibrils were intact. In the center of very strong lesions, where the collagen was hyalinized, birefringence was completely lost because of the complete disintegration of the fibrillar structure, which was visible under TEM. The transition zone toward normal cornea showed increased birefringence because the natural birefringence was largely preserved and enhanced by the increased Sirius-red uptake. Mechanical stretching between neighboring LTK lesions was manifested by increased birefringence. CONCLUSION: Sirius red offered an improved and simple histologic method for analyzing thermal collagen changes. It may contribute to a better understanding of the working mechanisms of LTK and improve analysis of thermal effects in corneal ablation.

Animals↗
PubMed1999

Extended resolution fluorescence microscopy.

Fluorescence microscopy is an essential tool of modern biology, but, like all forms of optical imaging, it is subject to physical limits on its resolving power. In recent years, several exciting techniques have been introduced to exceed these limits, including standing wave microscopy, 4Pi confocal microscopy, I5M and structured illumination microscopy. Several such techniques have been definitively demonstrated for the first time during the past year.

Animals↗
PubMedDate not supplied

Visualisation by confocal microscopy of traces on bullets and cartridge cases.

The capabilities of confocal scanning laser microscopy for the visualisation of marks on bullets and cartridge cases were investigated. Confocal microscopy provides solutions to important limitations of conventional comparison microscopy with grazing light incidence, as generally used for the examination of these marks. It is expected that confocal microscopy, thanks to its broad applicability within the field of firearms investigation and its capability of non-destructively gathering quantitative three-dimensional information, will lead to a more complete and objective forensic examination of bullets and cartridge cases.

Equipment Design↗
PubMed2002

Three-dimensional imaging of human skin and mucosa by two-photon laser scanning microscopy.

BACKGROUND: Various structural components of human skin biopsy specimens are difficult to visualize using conventional histologic approaches. METHODS: We used two-photon microscopy and advanced imaging software to render three-dimensional (3D) images of in situ nerves, blood vessels, and hair follicles labeled with various fluorescent markers. Archived frozen human skin biopsy specimens were cryosectioned up to 150 micro m in thickness and fluorescently stained with rhodamine- or fluorescein-labeled antibodies or lectins. Optical sections were collected by two-photon microscopy and the resulting data sets were analyzed in three dimensions using Voxx software. RESULTS: Reconstructed image volumes demonstrated the complex 3D morphology of nerves, blood vessels and adnexal structures in normal mucocutaneous tissue. CONCLUSION: Two-photon microscopy and Voxx rendering software allow for detailed 3D visualization of structures within human mucocutaneous biopsy specimens, as they appear in situ, and facilitate objective interpretation of variations in their morphology. These techniques may be used to investigate disorders involving cutaneous structures that are difficult to visualize by means of traditional microscopy.

Hair Follicle↗
PubMed2003

Plastic casts and confocal laser scanning microscopy applied to the observation of enamel tubules in the red Kangaroo (Macropus rufus).

Scanning electron microscopy for plastic casts and confocal laser scanning microscopy for Villanueva bone-stained ground sections were used together to observe enamel tubules in red kangaroo molars. Although the tubular structures such as terminals, bends, expansions, splits, divergences and rejoinings in this species were within the variations of marsupial species, their morphological characteristics were demonstrated with extremely clear and persuasive images. Thus, the combined observations of plastic casts by scanning electron microscopy and Villanueva bone-stain sections by confocal laser scanning microscopy were found to be of value for the investigation of enamel tubules and tubular structures in other hard tissues.

Acrylic Resins↗
PubMed1998

Phase-contrast microscopic studies using cinematographic techniques and scanning electron microscopy on IgE-mediated degranulation of cultured human mast cells.

BACKGROUND: Isolating human mast cells is a laborious procedure. Recently, cultured human mast cells raised from umbilical cord blood cells have become available. It is necessary to investigate whether IgE-mediated activation of these cells is mediated by exocytosis. OBJECTIVE: To verify IgE-mediated activation of these cultured human mast cells morphologically. METHODS: The mast cells were raised from human umbilical cord blood cells in the presence of stem cell factor and interleukin-6. IgE-sensitized cultured human mast cells were activated by anti-IgE, and morphological changes of the cells were examined under phase-contrast microscopy using cinematographic techniques and scanning electron microscopy. Histamine release from the cells was measured with high-performance liquid chromatography. RESULTS: Under the condition in which a significant histamine release was observed from the mast cells, phase-contrast microscopy showed that the cultured human mast cells became swollen and extruded granules. Scanning electron microscopy disclosed the extrusion of smooth and round bodies from pores formed on the activated mast cell surface. CONCLUSION: IgE-mediated histamine release from cultured human mast cells is accompanied by exocytosis morphologically, indicating that cultured human mast cells will help in studying the functional properties of human mast cells.

Cell Degranulation↗
PubMed2000

Carbon nanotube atomic force microscopy tips: direct growth by chemical vapor deposition and application to high-resolution imaging.

Carbon nanotubes are potentially ideal atomic force microscopy probes because they can have diameters as small as one nanometer, have robust mechanical properties, and can be specifically functionalized with chemical and biological probes at the tip ends. This communication describes methods for the direct growth of carbon nanotube tips by chemical vapor deposition (CVD) using ethylene and iron catalysts deposited on commercial silicon-cantilever-tip assemblies. Scanning electron microscopy and transmission electron microscopy measurements demonstrate that multiwalled nanotube and single-walled nanotube tips can be grown by predictable variations in the CVD growth conditions. Force-displacement measurements made on the tips show that they buckle elastically and have very small (</= 100 pN) nonspecific adhesion on mica surfaces in air. Analysis of images recorded on gold nanoparticle standards shows that these multi- and single-walled carbon nanotube tips have radii of curvature of 3-6 and 2-4 nm, respectively. Moreover, the nanotube tip radii determined from the nanoparticle images are consistent with those determined directly by transmission electron microscopy imaging of the nanotube ends. These molecular-scale CVD nanotube probes have been used to image isolated IgG and GroES proteins at high-resolution.

Animals↗
PubMedDate not supplied

Detection of VP-16-treated HL-60 cell apoptosis by TUNEL electron microscopy.

Apoptosis is essential to many physiological processes, including maturation of cells and the immune system. Deficient regulation of apoptosis may play an important role in many pathological conditions, such as autoimmunity, AIDS, and myelodysplastic syndrome. Several methods have been described to identify apoptotic cells. DNA strand breaks can be identified by labeling free 3'-OH termini with modified nucleotides by enzymatic reaction (TUNEL method). In this study, apoptosis was introduced in cultured HL-60 cells treated with VP-16, and the TUNEL method was adapted for electromicroscopy, called the EM TUNEL method. The results of the EM TUNEL method were compared with those of light microscopy and flow cytometry. Apoptotic cells following VP-16 (10 micrograms/mL) treatment were detected after 3 h by all methods (light microscopy, Erythrocin B, electron microscopy, flow cytometry, TUNEL, and EM TUNEL). EM TUNEL was the most sensitive method of detection, with a detection rate of 32%. Furthermore, EM TUNEL was more suitable for distinguishing cell lineage than light microscopy TUNEL. The results indicate that EM TUNEL can be used to detect apoptosis in bone marrow species in vivo.

Apoptosis↗