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At least 1,405 records · Page 78Linked to original sources
PubMed1988

Fluorescence microscopy of nuclear DNA in oocytes and zygotes during in vitro fertilization and development of early embryos in mice.

Fluorescence microscopic investigations of nuclear DNA stained with mithramycin or 4',6-diamidino-2-phenylindole (DAPI) were undertaken in oocytes and zygotes during in vitro fertilization (IVF) and subsequent early embryo development in mice. Mithramycin and DAPI bind stoichiometrically with G-C and A-T pairs of DNA, respectively. In the present study, it was possible to visualize the germinal vesicle, the nucleolus, and the chromosome in the oocyte. After IVF, the nuclei, male and female pronuclei, polar bodies, nucleolus-associated chromatins, and chromosomes were successively observed in the zygote, the blastomere during cleavage, and the blastocyst until the hatching stage. The formation of male and female pronuclei, the process of their fusion, the appearance of the chromosome, and the commencement of the first cleavage of the zygote to form two blastomeres were all clearly demonstrated. The number of blastomeres was easily counted in the morula in spite of the compaction of the blastomeres. The present study indicates that the methods reported herein are useful for the determination of exact functional stages of the oocyte and the zygote during IVF and early embryo development, and thus contributes to the advancement of reproductive and developmental biomedical research.

Animals↗
PubMed1995

Confocal fluorescence microscopy for studying thapsigargin-induced bivalent-cation entry into B cells.

We studied thapsigargin-induced bivalent-cation entry into antigen-specific B cells (TP67.21) with a confocal fluorescence microscope. Confocal fluorescence images of fluo-3-loaded B cells showed that thapsigargin-stimulated Ca2+ signals were transferred not only to the cytoplasm but also to the nucleus. In the absence of external Ca2+ ions, the free Ca2+ concentrations both in the cytosol and in the nucleus declined to basal levels by 5 min after addition of thapsigargin. However, subsequent addition of Ca2+ in the external medium made the fluo-3 (fura-2) fluorescence intensity rise, reflecting the fact that Ca2+ accumulated again in the nucleus as well as in the cytoplasm. Then, we added Ba2+ and Mn2+ instead of Ca2+, because Ba2+ and Mn2+ are known to enter via Ca2+ channels. The addition of Ba2+ and Mn2+ in the external medium quenched the fluo-3 fluorescence both in the nucleus and in the cytoplasm of B cells. This suggested the possibility that the increase in intranuclear Ca2+ after thapsigargin stimulation may come from the cytoplasm, not from the nuclear stores.

Aniline Compounds↗
PubMed2004

Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions.

The imaging of cellular dynamics in three dimensions using a standard microscope is severely limited due to the fact that only one focal plane can be imaged at a given point in time. Here we present a modification of the classical microscope design with which two or more focal planes can be imaged simultaneously. This is achieved by a modification of the emission pathway of a standard microscope. The efficacy of the design is shown by imaging bead samples and an FcRn-green fluorescent protein expressing tubule that leaves a sorting endosome and subsequently exocytoses at the plasma membrane.

Cell Line↗
PubMed1989

Three-dimensional imaging of neurons by confocal fluorescence microscopy.

The study of neuronal architecture by means of confocal laser microscopy is described. Optical serial sectioning has been performed on whole-mount specimens, and the resulting stacks of digitally recorded images have been processed with the help of a computer. Specimen preparation is described, as well as the instrument and its performance. It is shown that the limits in photometric quality are set by photon quantum noise. As both light absorption and scattering was low in the studied specimens, the maximum scanning depth was limited mainly by the working distance of the objectives. Compared with traditional methods, confocal microscopy in combination with digital image processing has the following advantages: (1) a truly three-dimensional (3-D) reconstruction is obtained, (2) the specimen remains intact, (3) a higher resolution can be obtained, (4) the process is automated and less time-consuming and (5) various kinds of data processing are possible.

Animals↗
PubMed1995

Reversible vacuolation of the transverse tubules of frog skeletal muscle: a confocal fluorescence microscopy study.

A confocal microscope was used to investigate the reversible vacuolation of frog skeletal muscle fibres produced by the efflux and entry of glycerol (80-100 mM). The formation, development and disappearance of vacuoles was observed in the fibres by staining simultaneously with two fluorescent membrane probes, RH414 and DiOC6(3). The styryl dye, RH414, stains only the plasmalemma and the membranes of the transverse tubules. In normal and glycerol-loaded fibres, RH414 revealed regular, narrow dotted bands located at the position of the Z-lines. Glycerol removal produced, within 2-10 min, many empty round vacuoles (0.4-1.5 microns in diameter) that were apparently anchored to the stained bands. Later on, individual vacuoles tended to enlarge and align into longitudinal chains of vacuoles. Neighbouring vacuoles that contacted each other fused to form large vacuoles up to several sarcomeres in length. Neither the T-tubules, nor the vacuoles, were stained by DiOC6(3). However, glycerol efflux was also accompanied by a redistribution of sarcoplasmic reticulum membranes and by changes in mitochondria that were revealed on staining the same fibres with the carbocyanine dye, DiOC6(3). The alterations in staining patterns revealed by RH414 and DiOC6(3) were completely reversible. Within 5-10 min after a second application of glycerol, the pattern of staining returned to normal with the exception of very bright, spots stained with RH414, which appeared in place of many but not all of the vacuoles, and probably correspond to the irregular nets of T-tubules observed under the electron microscope in such fibres. They are considered to be defects in regeneration of the T-system after vacuolation. The vacuolation/devacuolation cycle could be repeated several times following glycerol efflux and entry. The development and disappearance of vacuoles then mainly involved conversion of bright spots to large vacuoles and vice versa. Some possible mechanisms of vacuole formation and disappearance are discussed, and it is suggested that vacuolation of the T-system may be important in relation to regulating the volume of skeletal muscle cells.

Animals↗
PubMed1992

Evanescent interference patterns for fluorescence microscopy.

The increasing experimental use of total internal reflection/fluorescence photobleaching recovery has motivated a theoretical study of the spatial intensity profiles generated by two interfering evanescent waves. The interference patterns generated by evanescent waves differ considerably from those generated by plane waves in a homogenous medium because evanescent waves are not transverse and because the evanescent propagation number depends on the incidence angle of the totally internally reflected light. The periodicity and contrast of the evanescent interference patterns under various conditions are calculated; these parameters depend on the intensities, polarizations, and incidence angles of the two incident beams, as well as the refractive indices of the two media that form the planar interface where total internal reflection occurs. The derived intensity profiles are used to develop expressions for the shapes of fluorescence photobleaching recovery curves when evanescent interference patterns are used for fluorescence excitation and bleaching. The calculations also suggest that colliding beam experiments may confirm theoretically predicted evanescent field polarizations.

Binding Sites↗
PubMed2005

Morphological and growth altering effects of Cisplatin in C. albicans using fluorescence microscopy.

Changes in morphology and growth curve of Candida albicans in response to treatment by Cisplatin has been studied using fluorescence staining with ethidium bromide. Treatment with Cisplatin was found to markedly inhibit hyphae and ovoid growth as revealed by ethidium bromide staining of drug treated cells. These changes were concomitant with inhibitory effects on the growth curve with respect to untreated cells. Presence of Cisplatin not only caused suppression in the limiting values in the growth curve, but also caused a slight left shift in the EC50 values. Some of the ovoid cells undergoing poisoning with cisplatin were found to be unusually enlarged before undergoing their natural fate thus suggesting formation of similar cytotoxic end products with DNA.

Candida albicans↗
PubMed1994

Cytosolic [Ca2+] measurements in endothelium of rabbit cardiac valves using imaging fluorescence microscopy.

Cytosolic Ca2+ plays a critical role in the secretion of endothelium-derived factors. A new preparation that allows fluorescence imaging of intracellular free Ca2+ concentration ([Ca2+]i) in endothelial cells of rabbit cardiac valves is described. Electron micrographs of the valves revealed no underlying smooth muscle cells that might influence endothelial cell responses or contribute to [Ca2+]i signaling. The valve leaflets, which were < 100 microns in diameter, were visualized using a specially designed chamber and a long working distance fluorescence objective. The semilunar valves (pulmonary and aortic) responded to endothelium-dependent vasodilators, including acetylcholine, with an increase in [Ca2+]i. Synchronized [Ca2+]i transients were observed in the endothelial monolayer in response to agonist stimulation in K(+)-free solutions. The ability to monitor changes in [Ca2+]i in a native endothelial monolayer provides a more realistic assessment of stimulus-response coupling within individual cells and communication between cells of native endothelium. In addition, this preparation affords an opportunity for comparative studies of endothelium-related pathophysiologies, which can be induced experimentally in animal models.

Acetylcholine↗
PubMed1980

Infrared fluorescence microscopy of stained tissues: principles and technic.

Infrared photomicrography was used extensively from 1927 to the 1940's, but received little attention during the last decades. However, studies of infrared fluorescence of stained sections could not be found in the accessible literature. Ramsley (1968) published quantitative data on infrared fluorescence of approximately 250 dyes bound to textile fibers. The intensity of infrared fluorescence of many dyes varied widely with the substrate. It was therefore deemed of interest to determine whether or not similar differences in infrared fluorescence may occur when dyes are bound to histochemically distinct tissue structures. Myofibrils and collagens stained with triarylmethane dyes were chosen as test objects. Kodak infrared cut-off filter No. 301 and Wratten filter #16 were used as exciter filters to remove infrared and UV-blue and the light of a xenon lamp. Wratten filter #70 and #89B were employed as barrier filters. Infrared radiation was recorded with Kodak Ektachrome infrared film. To facilitate correlation of infrared fluorescence patterns with visible images, tissues were photographed also with conventional color film. Stained myofibrils, e.g. in myoepithelium, smooth and striated muscle emitted strong infrared fluorescence; collagen showed little or no fluorescence. Barrier filter Wratten #70 permitted simultaneous demonstration of infrared fluorescence and of non-fluorescent structures and thus facilitated histopathological studies. Preliminary findings indicate decrease or loss of infrared fluorescence of stained muscle fibers in various lesions, e.g. myocardial infarction, Duchenne-type muscular dystrophy.

Connective Tissue↗
PubMed1978

[Remarks on fluorescence microscopy of cerebrospinal fluid cells (author's transl)].

Fluorescencemicroscopic investigations of the liquor cells were carried out by acriclinorange, trypaflavea, the fluorescence component of anilinblue and morin in connection with thiacinred. After that it will be reported about microscopphotometrical measure ments and examinations of immunfluorescence and emphasized the importance of these methods for cytochemistry of the cerebrospinal fluid.

Antigen-Antibody Reactions↗
PubMed2004

Direct fluorochrome labeling of phage display library clones for studying binding specificities: applications in flow cytometry and fluorescence microscopy.

Phage display technology is increasingly employed to identify high-affinity peptides and single-chain antibodies with binding specificities for a diversity of target types. The analysis of phage-binding sensitivity and specificity typically employs directly labeled secondary antiphage antibodies and potentially tertiary labels, such as fluorochromes and enzymes, when biotinylated antibodies are used. However, secondary or tertiary reagents may not be feasible or desirable for some target types and applications. Here, we present a simple approach for directly labeling phage clones with two common amine-reactive fluorochromes. We show that these fluorochromes label the pVIII major coat protein and that the binding selectivity of peptides displayed on the pIII protein of several well-characterized phage clones is maintained in flow cytometric analysis and immunofluorescence microscopy. Uniquely, such labeled phage, in part, represent self-propagating reagents because conjugation does not impair the ability to efficiently reproduce in bacteria, although relabeling with fluorochrome would be necessary. Our data suggest that primary labeled phage clones may be used similarly to primary antibody conjugates.

Animals↗
PubMed1985

Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths.

Free Ca2+ concentrations in the cytosol of individual small cells can be recorded with a new fluorescent Ca2+ indicator, "fura-2", and a fluorescence microscope modified to chop rapidly between two wavelengths of excitation. Both fura-2 and its Ca2+ complex fluoresce strongly, but their excitation peaks differ in wavelength. Alternation between the two preferred wavelengths allows assessment of the ratio of Ca2+-bound dye to free dye and hence cytosolic free Ca2+. This ratio measurement largely cancels out the effects of cell thickness, dye content, or instrumental efficiency, uncertainties that can jeopardize measurements at single wavelengths. We describe instrumentation that supplies rapidly alternating excitation wavelengths to either a standard cuvet or a fluorescence microscope. Its use is illustrated by experiments showing changes in cytosolic [Ca2+] accompanying activation of human platelets in suspension or single mouse thymocytes on the microscope.

Animals↗
PubMed1995

A new fluorescence microscopy for tomographic observation of microcirculation by using dual-beam slit laser illumination.

By designing a new epi-illumination system with dual slit laser beams, we have developed a fluorescence microscope for tomographic observations of in vivo microcirculation. Two beams of an Argon ion laser were converted into thin slit beams of thickness 28 to 60 microns by optical lenses and were illuminated onto tissue to intersect at the focal plane of the objective. The fluorescent light emitted from a FITC tracer by this cross-illumination was picked up by the microscope through a filter, with no "out of focus" noise from the tracer above and below the crossing zone. The results of in vitro experiments by using glass micropipettes (20-80 microns i.d.) filled with FITC-dextran and skeletal muscle tissue certified uniform tracer excitation in the crossing zone and the augmentation of the beam thickness due to light scattering in the tissue to be 1.7 and 2.5 times at depths of 100 and 200 microns from the surface, respectively. In vivo tests in the rabbit tenuissimus muscle revealed that this system can realize microvasculature tomography, with adequate vertical zone selectivity and spatial resolution to reconstruct its three-dimensional mapping, and long-term monitoring of tracer leakage from a single capillary to the surrounding tissue, with sufficient quantitative reliability to determine the capillary permeability and its heterogeneity along the channel.

Animals↗