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At least 1,513 records · Page 84Linked to original sources
PubMed2004

Automated microscopy identifies estrogen receptor subdomains with large-scale chromatin structure unfolding activity.

BACKGROUND: Recently, several transcription factors were found to possess large-scale chromatin unfolding activity; these include the VP16 acidic activation domain, BRCA1, E2F1, p53, and the glucocorticoid and estrogen steroid receptors. In these studies, proteins were fluorescently labeled and targeted to a multimerized array of DNA sequences in mammalian cultured cells, and changes in the appearance and/or size of the array were observed. This type of experiment is impeded by the low throughput of traditional microscopy. METHODS: We report the application of automated microscopy to provide unattended, rapid, quantitative measurements of fluorescently labeled chromosome regions. RESULTS: The automated image collection routine produced results comparable to results previously obtained by manual methods and was significantly faster. Using this approach, we identified two subdomains within the E domain of estrogen receptor alpha capable of inducing large-scale chromatin decondensation. CONCLUSIONS: This work confirms that, like BRCA1, the activation function 2 region of the estrogen receptor has more than one distinct chromatin unfolding domain. In addition, we demonstrate the feasibility of using automated microscopy as a high-throughput screen for identifying modulators of large-scale chromatin folding. The Supplementary Material referred to in this article can be found at the CYTO Part A website (http://www.interscience.wiley.com/jpages/0196-4763/suppmat/v58A.html)

Animals↗
PubMed2004

Scanning fluorescent microscopy is an alternative for quantitative fluorescent cell analysis.

BACKGROUND: Fluorescent measurements on cells are performed today with FCM and laser scanning cytometry. The scientific community dealing with quantitative cell analysis would benefit from the development of a new digital multichannel and virtual microscopy based scanning fluorescent microscopy technology and from its evaluation on routine standardized fluorescent beads and clinical specimens. METHODS: We applied a commercial motorized fluorescent microscope system. The scanning was done at 20 x (0.5 NA) magnification, on three channels (Rhodamine, FITC, Hoechst). The SFM (scanning fluorescent microscopy) software included the following features: scanning area, exposure time, and channel definition, autofocused scanning, densitometric and morphometric cellular feature determination, gating on scatterplots and frequency histograms, and preparation of galleries of the gated cells. For the calibration and standardization Immuno-Brite beads were used. RESULTS: With application of shading compensation, the CV of fluorescence of the beads decreased from 24.3% to 3.9%. Standard JPEG image compression until 1:150 resulted in no significant change. The change of focus influenced the CV significantly only after +/-5 microm error. CONCLUSIONS: SFM is a valuable method for the evaluation of fluorescently labeled cells.

Fluorescent Dyes↗
PubMed2004

Microscopy-based multicolor tissue cytometry at the single-cell level.

Cytomics is a novel perspective from which to look at life. As with genomics and proteomics before, this discipline requires novel and innovative techniques and technologies to focus on its substrate of research--the cytome. With cytomics being the discipline that analyzes cellular systems and their interdependencies, advanced microscopy represents a key technology in cytomics research. Yet, conventional microscopy-based investigations, i.e., "look and conclude" analyses, do not meet the major cytomics criteria of 1) relating multiple parameters to each other, 2) within large populations of cells, 3) on a single-cell basis, and 4) in a quantitative and observer-independent manner. However, emerging improvements in the fields of fluorophore technology, sensitive fluorescence detection devices, and sophisticated image analysis procedures, are important and necessary steps into the cytomics era. Tissue represents an important class of cytomes, hence tissue cytometry--on the single cell level--can be expected to become an important cytomics technology. In this report, the techniques and technologies of microscopy-based multicolor tissue cytometry (MMTC) are outlined and applications are discussed, including the phenotypic characterization of tissue infiltrating leukocytes, in situ quantification of proliferation markers and tumor suppressors, and in situ quantification of apoptosis.

Apoptosis↗
PubMed1988

Fluorescence of Blastomyces and other fungi in Papanicolaou-stained pulmonary cytology preparations: comparison with light microscopy.

In this study, 152 Papanicolaou-stained pulmonary cytologic smears from 15 known cases of pulmonary fungal infection were randomly mixed with 194 control pulmonary smears. All slides were examined for fungi by three observers, first by light microscopy and then by fluorescent microscopy. The results of the light and fluorescent microscopy were compared. It was concluded that when both methods were used for fungal detection, the yield of positive results was higher.

Blastomyces↗
PubMed1994

Matrix crystals in cytologic urine specimens: observations on their mineral composition by energy dispersive X-ray microanalysis and morphologic scanning electron microscopy.

Crystals consisting by light microscopy of organic matrix (matrix crystals) encountered in cytologic urine specimens of 8 patients were examined for mineral phase components by scanning electron microscopy with energy dispersive X-ray microanalysis (SEM-EDX) and by morphologic scanning electron microscopy (SEM) performed separately in four of the eight cases. Whenever possible (three cases) mineralized crystals present in these specimens were examined separately by SEM-EDX for comparison of mineral phase composition with that of the corresponding matrix forms. Although by SEM-EDX components of matrix, glass and slide preparation media interfere with the precise estimation of the mineral phase components, the results of this method supported by the SEM morphology suggest that crystals consisting of organic matrix include a mineral phase, the lattice structure of which provides them from the early stages of formation with the characteristic morphology of the fully mineralized forms. This also suggests that organic matrix plays a role in the nucleation of minerals during the formation of certain urinary crystals.

Adult↗
PubMedDate not supplied

Electron microscopy in the diagnosis of head and neck tumors.

Six patients with malignant head and neck tumors are shown to have required electron microscopy for accurate diagnosis. In all of these tumors, there were ultrastructural features of cytodifferentiation that were not discernible by light microscopy, such as neurosecretory granules, desmosomes, cytoplasmic processes, tonofibrils, and myofilaments. Electron microscopy is helpful in the differential diagnosis of tumors in general, but its effectiveness is particularly apparent in small-cell "undifferentiated" tumors such as neuroblastoma, rhabdomyosarcoma, Ewing's sarcoma, undifferentiated squamous-cell carcinoma of the lymphoepithelioma type, and malignant lymphoma. It has also been helpful in the identification of amelanotic melanoma and spindle-cell carcinoma.

Adult↗
PubMed1998

Magnetic resonance microscopy and histopathology: comparative approach of bromobenzene-induced hepatotoxicity in the rat.

The development of magnetic resonance (MR) microscopy has provided new approaches to histology and histopathology. Recent work has shown the promise of increased sensitivity in animal models of chemically induced hepatotoxicity. However, the field is so new that there is little experience to relate changes seen in MR micrographs to the more traditional optical images stained with hematoxylin and eosin. This work compares the sensitivity and reproducibility of MR microscopy with conventional histopathology in detecting bromobenzene-induced hepatotoxicity in the rat. A time-course study was undertaken to provide a range of histopathologies. Specimens were studied at 24, 48, 72, and 96 hours after exposure to 10% of the median lethal dose of bromobenzene. Using 4 animals per group (a total of 32 rats) added statistical significance to the study and defined a range of interanimal variability over 96 hours. This work shows that MR microscopy, besides being nondestructive and three-dimensional, is at least as sensitive as conventional hematoxylin-eosin staining in detecting bromobenzene-induced centrilobular lesions and recovery of the hepatocellular architecture in the rat. This study further suggests that, as we begin to understand the underlying mechanisms of contrast in MR histology, MR may, in fact, supply even higher specificity than more traditional studies: variations were observed in MR images of treated livers at a given time point that could be not be differentiated based on the grading of necrosis and inflammation on hematoxylin-eosin-stained sections.

Animals↗
PubMed2000

Microstructural evolution of lipid aggregates in nucleating model and human biles visualized by cryogenic transmission electron microscopy.

Obtaining reliable information on the physical state and ultrastructure of bile is difficult because of its mixed aqueous-lipid composition and thermodynamic metastability. We have used time-lapse cryogenic transmission electron microscopy (cryo-TEM) combined with video-enhanced light microscopy (VELM) to study microstructural evolution in nucleating bile. A well-characterized model bile and gallbladder biles from cholesterol and pigment gallstone patients were studied sequentially during cholesterol nucleation and precipitation. In model bile, cholesterol crystallization was preceded by the appearance of the following distinct microstructures: spheroidal micelles (3-5 nm), discoidal membrane patches (50-150 nm) often in multiple layers (2-10), discs (50-100 nm), and unilamellar (50-200 nm) and larger multilamellar vesicles (MLVs). The membrane patches and discs appeared to be short-lived intermediates in a micelle-to-vesicle transition. Vesicular structures formed by growth and closure of patches as well as by budding off from vesicles with fewer bilayers. MLVs became more abundant, uniform, and concentric as a function of time. In native bile, all the above microstructures, except discoidal membrane patches, were observed. However, native MLVs were more uniform and concentric from the beginning. When cholesterol crystals appeared by light microscopy, MLVs were always detected by cryo-TEM. Edges of early cholesterol crystals were lined up with micelles and MLVs in a way suggesting an active role in feeding crystal growth from these microstructures. These findings, for the first time documented by cryo-TEM in human bile, provide a microstructural framework that can serve as a basis for investigation of specific factors that influence biliary cholesterol nucleation and crystal formation.

Bile↗
PubMed1985

Electron microscopy of SV40 DNA cross-linked by anti-Z DNA IgG.

Electron microscopy has revealed the specific binding of bivalent anti-Z DNA immunoglobulin G (IgG) to different sites on supercoiled Form I SV40 DNA. The anti-Z IgG links together left-handed regions located within individual or on multiple SV40 DNA molecules at the superhelix density obtained upon extraction. Velocity sedimentation, electrophoresis, and electron microscopy all show that two or more Z DNA sites in the SV40 genome can be intermolecularly cross-linked with bivalent IgG into high mol. wt. complexes. The formation and stability of the intermolecular antibody-DNA complexes are dependent on DNA superhelix density, as judged by three criteria: (1) relaxed circular (Form II) DNA does not react; (2) release of torsional stress by intercalation of 0.25 microM ethidium bromide removes the antibody; and (3) linearization with specific restriction endonucleases reverses antibody binding and DNA cross-linking. Non-immune IgG does not bind to negatively supercoiled SV40 Form I DNA, nor are complexes observed in the presence of competitive synthetic polynucleotides constitutively in the left-handed Z conformation; B DNA has no effect. Using various restriction endonucleases, three major sites of anti-Z IgG binding have been mapped by electron microscopy to the 300-bp region containing nucleotide sequences controlling SV40 gene expression. A limited number of minor sites may also exist (at the extracted superhelix density).

Antibodies↗
PubMed1994

Confocal laser scanning microscopy (CLSM) for the study of collagen sponge microstructure.

This study uses confocal laser scanning microscopy (CLSM) to assess the microstructure of collagen sponges providing an accurate quantification of porosity under conditions similar to those experienced by cells growing on the sponges during culture. CLSM offers several advantages over scanning electron microscopy (SEM) and conventional optical microscopy for this kind of study, the most important of which is probably the absence of artifacts associated with the extensive preparation of samples required for the latter two methods. When the "pan-side" surface of collagen sponges was studied, it was found that the pore sizes increased with increasing depth into the sponge. Collagen sponges frozen in a -70 degrees C freezer had a more open structure than ones frozen on the stage of a tissue dryer. These different pore sizes are thought to reflect different freezing rates in the samples.

Animals↗
PubMed2005

Analytical technique for quantification of selected resorbable calcium phosphate bone void fillers with the use of polarized-light microscopy.

Synthetic calcium phosphate bone void fillers promote varying rates of bone formation and material resorption depending on chemistry, porosity, pore structure, and implant site. The objective of this study was to quantify the resorption of a novel ultraporous beta-tricalcium phosphate cancellous bone void filler with simultaneous quantification of bone formation in a canine humerus model. Potential measurement error involved in conventional histomorphometry using Von Kossa stains inspired the development of a new technique. This technique utilizes bright-field and polarized-light microscopy in conjunction with image analysis software, allowing more accurate histomorphometry. This technique was validated with two separate controlled experiments. Scanning electron microscopy further supported the results. The findings suggest that the use of polarized-light microscopy combined with image analysis software can be an effective tool in simultaneously quantifying calcium phosphate resorption and bone formation.

Animals↗
PubMed1994

Automated three-dimensional characterization of osteoclastic resorption lacunae by stereoscopic scanning electron microscopy.

The use of stereoscopic scanning electron microscopy to analyze quantitatively the topography of excavations made by osteoclasts in slices of devitalized cortical bone was evaluated. Using this innovative technique, the need mechanically to tilt the specimen stage to gather three-dimensional information is obviated by instead tilting the electron beam both to produce real-time stereo pairs and to gather measurement data. Based on the comparison of two images of microscopic areas, cross-correlation is used to determine the image shift between the features in the stereo pair. This information is then used dynamically to correct the height of the tilt axis and lens focus in a feedback loop, generating a quantitative measurement of height difference. With this approach, relative heights of individual points, line profiles, area topography maps, and three-dimensional reconstructions of excavations were obtained rapidly and at high resolution. This approach combines the advantage in topographic data acquisition of confocal microscopy with the advantages of the increased resolution and focal depth of scanning electron microscopy (SEM). The technique should facilitate not only the topographic analysis of osteoclastic excavations in bone slices at high resolution but also the three-dimensional analysis of the structure of bone tissue.

Bone Resorption↗
PubMed2002

Phosphorescent metalloporphyrins as labels in time-resolved luminescence microscopy: effect of mounting on emission intensity.

In this study, we present an investigation of the effects of mounting media on the phosphorescence of metalloporphyrin stained microscopy samples. The samples were: (1) Platinum(II) coproporphyrin (=PtCP) stained porous Sephadex beads; (2) compact polystyrene microspheres coated with IgG-PtCP conjugate; and (3) immunocytochemically labeled human peripheral blood neutrophils. The human neutrophils in a mixed leukocyte population were fixed, permeabilized, and then immunolabeled with PtCP conjugate of monoclonal mouse IgG directed to the intracellular antigen myeloperoxidase. The samples were mounted in twelve different mounting media and studied with quantitative time-resolved luminescence imaging microscopy with respect to the intensity and stability of the phosphorescence signal. The results indicate that microscopy samples stained with PtCP exhibit the brightest phosphorescence emission in non-mounted form or when mounted in non-aqueous permanent mounting media.

Animals↗
PubMed1988

Reflection high resolution analytical electron microscopy: a technique for studying crystal surfaces.

Reflection electron microscopy (REM), reflection high energy electron diffraction (RHEED), reflection electron energy-loss spectroscopy (REELS), and energy dispersion x-ray spectroscopy (EDX) have been comprehensively used as a technique, termed reflection high resolution analytical electron microscopy (RHRAEM), for studying the structures of the bulk crystal GaAs (110) surfaces by transmission electron microscopy (TEM). The simultaneous observations of surface topography imaging, the surface diffraction mechanism with RHEED, surface atomic inner-shell excitations with REELS, and surface chemical compositions with EDX provide a systematic description of the atomic structure and chemical structure of the surface. The surface channelling effect has been observed in GaAs (110) with REELS, which may provide a basis for localizing surface foreign atoms with ALCHEMI. The theoretically predicted surface-resonance wave has been observed directly in the RHEED pattern; the surface-captured Bragg reflection wave have been identified. It is shown that surface chemical compositions can be determined by analyzing the EDX spectra obtained in the REM case. Finally, the surface monolayer resonance characteristic of the RHRAEM has been confirmed by calculations with dynamical RHEED theory.

Arsenic↗
PubMed1989

The research center for ultra-high voltage electron microscopy at Osaka University.

High-voltage electron microscopy has shown itself advantageous for the study of natural science, including biology, but especially for materials science. The most important advantage for materials science is for in situ experiments about the detailed processes of the phenomena that occur in bulk materials. The present paper is mainly concerned with several types of in situ experiments that have been carried out in the Research Center for Ultra-High Voltage Electron Microscopy, Osaka University. The following subjects have been studied: a) fundamental problems, such as the conditions necessary for in situ experiments, functional features of specimen treatment devices, and the effects of electron irradiation; b) the dislocation behavior of crystals under various conditions; c) high-temperature behavior of refractory materials, mainly ceramic composites; d) new applications of electron irradiation effects, such as amorphization of crystalline materials and electron-irradiation-induced foreign-atom implantation; e) environment-matter interaction, mainly chemical amorphization of alloys; and f) future trends of the in situ experiment, such as combinations with Auger valency electron spectroscopy and high-resolution electron microscopy.

Crystallization↗
PubMed1991

Confocal fluorescence microscopy and three-dimensional reconstruction.

Several recent technological advances have considerably improved the field of confocal fluorescence microscopy. Improvements in confocal microscope design, new fluorescent probes and indicators, more sensitive imaging devices, and computer advances which allow for data manipulation and storage provide a convenient method to acquire complex three-dimensional (3-D) architectural details which previously were difficult or impossible to obtain from biological specimens. Applications of the laser scanning and tandem scanning confocal microscopes offer the potential for gaining powerful insights into the complex relationship of cellular structure and function. Confocal microscopy generates optical sections free from out-of-focus blur. With the development of new visualization tools to render and display complex 3-D data, a set of optical sections taken at different focal planes can be three-dimensionally reconstructed to create an animated sequence which can reveal latent features of the specimen. The combination of confocal microscopy and 3-D reconstruction provides a powerful new imaging tool to advance knowledge about structural and functional cellular properties as they occur dynamically in three dimensions.

Animals↗
PubMed1991

Cryo-electron microscopy of vitrified specimens: an approach to the study of bulk specimens.

We are using and developing cryo-electron microscopy of vitrified specimens. Our main interests concern the structure of muscle and muscular components. Micrographs which generally contain periodic features are analyzed by numerical image processing methods. To detect artifacts induced by the electron microscopy techniques, we correlate our results to those obtained by X-ray diffraction. In this paper, we describe our approach to the study of bulk specimens. Vitrification of such specimens is assessed by cryo-X-ray diffraction. Microscopy is done on cryo-substituted specimens.

Animals↗
PubMed1993

Aerosolized fluorescent microspheres detected in the lung using confocal scanning laser microscopy.

Aerosolized fluorescent microspheres were used to study particle deposition in site-specific regions of the lung with confocal laser scanning microscopy. A nebulizer was used to aerosolize microspheres followed by passage through a heated discharging column to reduce static charge and to remove water surrounding each microsphere. Precoating of microspheres with albumin helped to minimize displacement during vascular fixation of the lungs. Confocal laser microscopy facilitated visualization of microspheres throughout the bronchial tree, ducts, and alveoli of the lungs. The use of fluorescent microspheres and confocal laser imaging provided distinct advantages compared with other methods to study lung particle deposition due to (1) the generation of single microspheres of uniform size by nebulization, (2) easy detection of microspheres in large slabs of microdissected lung tissues, (3) excellent resolution of tissue surfaces and microspheres for an infinite number of orientations and planes of section, and (4) the ability to visualize microspheres below fluid lining layers and on surfaces that could not easily be done by other methods of microscopy.

Aerosols↗