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At least 1,531 records · Page 85Linked to original sources
PubMed2000

Giant phospholipid vesicles: comparison among the whole lipid sample characteristics using different preparation methods: a two photon fluorescence microscopy study.

Several methods for the preparation of giant unilamellar vesicles (GUVs) using synthetic phosphatidylcholine phospholipids were evaluated. We compared the physical characteristics--in terms of lamellarity and morphology--of the whole lipid sample for each different lipid preparation using the sectioning capability of the two-photon excitation fluorescence microscope. From the evaluation of the entire lipid sample we determined that vesicle size, internal shape and shell thickness distributions depend on the vesicle's preparation method. Our results show that the preparation of giant unilamellar vesicles by the application of external electric fields offers several advantages among the other methods tested here. Using this method a high yield (approximately 95%) of giant unilamellar vesicles with a narrow size distribution was obtained. Independently of the preparation method, some lipid structures, which are held together by lipid tethers, were identified and resolved. These particular lipid structures show shell thickness and size heterogeneity. Labeling the lipid samples with 6-lauroyl-2-(N,N-dimethylamino)naphtalene (LAURDAN) and using the LAURDAN generalized polarization function we show that the lipid packing in these tethers or tubes is similar to those found in the phospholipid vesicles. The fact that both vesicles and tethers are found in the lipid preparations indicates similar stability between these structures.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1997

Dual image of myosin and actin in human neutrophils during movement by fluorescence microscopy.

It is well known that myosin and actin play important roles as a motile apparatus in cells, and as a cytoskeleton of cell structure in non-muscle cells. The purpose of this study was simultaneously to demonstrate the intracellular dynamics of myosin and actin in human neutrophils during movement. We designed a double-fluorescence labelling procedure utilising fluorescein isothiocyanate and rhodamine. Myosin was labelled with the green-fluorescence, F-actin was labelled with the red-fluorescence and the coexisting myosin and F-actin were labelled with yellow-fluorescence. We obtained the dual image of myosin and F-actin in human neutrophils during movement by this procedure through a conventional fluorescence microscope. Dual image can interpret the relationship between myosin and actin in human neutrophils during movement, however, further studies are required to elucidate the contractile mechanism in such cells.

Actins↗
PubMed2006

Fluorescence microscopy of NaCl-stressed, elongated Salmonella and Listeria cells reveals the presence of septa in filaments.

The cell morphology of Salmonella enteritidis and Listeria monocytogenes after the application of stress was examined. Cells were stressed by plating the bacteria on Tryptone Soya Agar (TSA) plates, with 5-10% NaCl. The plates were subsequently incubated for 6 days at 25 degrees C. Finally, the cells were harvested and subjected to different fluorescent probes in order to visualize the possible presence of septa in elongated cells. Use of the stain 4',6-Diamidino-2-phenylindole (DAPI), which is a blue fluorescent nucleic acid stain that preferentially stains double-stranded DNA, showed clearly the presence of dark spots, probably cellular partitions where no nucleic acids were present, in both Salmonella and Listeria cells. Another stain, FM 4-64, a lipophilic styryl dye for red staining of the inner membrane, showed the presence of highly fluorescent spots in Listeria cells, probably indicating the presence of membranes. For Salmonella, however, FM 4-64 was not successful in revealing septa in filaments. Double staining applied to elongated Listeria cells showed areas with high fluorescence in DAPI-staining (DNA-rich spots) which contained low fluorescence in FM 4-64-staining (membrane spots) and vice versa, which is a confirmation that the elongated cells are indeed composed of several normal size cells.

Fluorescent Dyes↗
PubMed1995

Cytokine receptor expression in human lymphoid tissue: analysis by fluorescence microscopy.

A highly-sensitive flourescence method, capable of detecting cytokine receptors present at low concentrations (around 100 molecules per cell) by flow cytometry, was adapted for use on tissue sections. This method was used to examine the expression of several cytokine receptors in lymphoid tissues. IL-2 receptors were distributed broadly, with higher concentrations in T cell areas. IL-1 receptor Type 1 was detected in T cell areas and in the follicular mantle, and was strongly expressed on vascular endothelium. IL-6 receptor was found at very low concentration, both within and outside germinal centres. The gp 130 molecule, which is involved in the functional receptor complex for IL-6 and several other cytokines, was present at higher concentrations, particularly in the germinal centre. Analysis of receptor expression in secondary lymphoid tissue provides evidence bearing on the physiological roles of cytokines, as these tissues contain cells at various stages of physiological activation located in well-defined functional zones.

Antibodies, Monoclonal↗
PubMed1995

Cell and tissue distribution of synthetic oligonucleotides in healthy and tumor-bearing nude mice. An autoradiographic, immunohistological, and direct fluorescence microscopy study.

Antisense oligonucleotides have the ability to inhibit individual gene expression in the potential treatment of cancer and viral diseases. However, the way parenterally administered oligonucleotides distribute themselves into healthy tissues or tumors is poorly understood. In this study, the cell and tissue distribution of two modified or unmodified phosphodiester pentadeca-beta-oligonucleotides intravenously administered to healthy or tumor-bearing nude mice was assessed by autoradiography as well as by direct fluorescence and immunoenzymatic histological methods. Resistance of oligonucleotides to degradation by nuclease activity was previously studied in vitro. Using these methods we were able to show the following: 1) within minutes, oligonucleotides permeate all cells and tissues with the exceptions of erythrocytes and intervertebral discs; 2) cell and tissue distribution does not depend on the sequence of the given oligonucleotide; 3) concentration of oligonucleotides is higher within the connective tissue cells than in the interstitial matrix; 4) after uptake, oligomers partition throughout all of the cellular compartments, including at the highest intracellular concentrations in the nuclei; 5) oligonucleotides penetrate easily the tumor cell compartments, oligonucleotide diffusion being unimpeded by the extracellular matrix.

Animals↗
PubMed2005

Classification of nerve cells from substantia nigra of patients with Parkinson's disease and amyotrophic lateral sclerosis with the use of X-ray fluorescence microscopy and multivariate methods.

The causes of Parkinson's disease and amyotrophic lateral sclerosis are still not known, but there is evidence that metal ions can be involved in processes leading to degeneration and atrophy of neurons in the case of these two neurodegenerative disorders. A synchrotron microbeam X-ray fluorescence technique was applied for topographic and quantitative analyses of selected elements on central nervous system tissue. The thin slices of brain were measured on the undulator beamline ID 22 at the European Synchrotron Radiation Facility in Grenoble, France. The polychromatic beam with the dimension of 5 microm x 2 microm (horizontal x vertical) was used in measurements. Tissues of substantia nigra representing Parkinson's disease, amyotrophic lateral sclerosis, and the control case were scanned. The results obtained indicated that accumulation of some elements depends on the case that the substantia nigra represents. Some variability in the elemental distribution for a given case was noticed as well. To investigate if present differences in the elemental accumulation between analyzed cases are statistically significant, multivariate methods were used. Cluster and discriminant analyses confirmed the significance of the differences in elemental accumulation in biological structures representing the examined cases. The methods used let us classify these structures in separate groups and determine elements, which play the greatest role in the differentiation of the biological structures for each case.

Amyotrophic Lateral Sclerosis↗
PubMed1991

Single cell fusion events induced by influenza hemagglutinin: studies with rapid-flow, quantitative fluorescence microscopy.

Fusion of individual human erythrocytes to fibroblasts expressing the influenza virus hemagglutinin Cells were attached to coverslips fitted in a specially designed flow chamber mounted on a microscope stage, and fusion was triggered by rapid acidification to pH less than 5.2. Fusion between single cell pairs was monitored by a fluorescence increase due to redistribution of fluorescent dyes between either membrane or cytoplasmic compartments of fusing cells. The single cell fusion events were broadly heterogenous in lag times, rise times, and overall shape of the curves. Lag times obtained with a water-soluble dye were within the range obtained with a water-soluble dye were within the range obtained with the membrane-bound fluorophores, (10-160 s). Fusion was both all-or-nothing and irreversible, in that once dye redistribution in any cell commenced, it completed, regardless of pH. Short pulses of pH 4.9 for 6-10 s led to about half of the cell pairs fusing, but pulses greater than 14 s were as effective as constant low pH. Pulses that were too short to trigger fusion did not partially activate nor deactivate the fusion process, as shown by the ability of a second acidification to cause fusion of the same cells, with similar lag times. These results indicate that the overall hemagglutinin-mediated fusion process is composed of at least two stages, one required for commitment of the hemagglutinin to a fusogenic state that is pH-dependent and a maturation stage that is pH-independent.

Animals↗
PubMed2004

In vivo fluorescence microscopy of neuronal activity in three dimensions by use of voltage-sensitive dyes.

We report in vivo imaging of neuronal electrical activity from superficial layers of the mouse barrel cortex. The measurements have approximately 16-microm spatial and 3-ms temporal resolution and reach depths of 150 microm below the cortical surface. The depth-dependent differential-fluorescence optical sections of activity are consistent with known cortical architecture and represent an important step toward in vivo measurement of functioning complex neural networks. Our observations employ a custom gradient-index lens probe and voltage-sensitive dye fluorescence; the use of epi-illumination rather than dark-field illumination provides the dramatic signal-to-noise improvement necessary for fast three-dimensional imaging.

Animals↗
PubMed1992

Distribution of actin filament lengths measured by fluorescence microscopy.

We analyzed the distribution of actin filament lengths by optical microscopy (OM). OM avoids possible alterations in the size or structure of actin filaments occurring during sample preparation for electron microscopy (EM). Images of F-actin labeled with tetramethylrhodamine isothiocyanate (TRITC)-phalloidin were analyzed for both size distribution and flexibility. In the standard buffer [25 mM potassium acetate, 4 mM MgSO4, 25 mM tris(hydroxymethyl)aminomethane acetate, pH 7.5, 20 mM beta-mercaptoethanol] filaments did not aggregate into bundles and remained stable at nanomolar concentrations for at least 1 h. At the same concentration, actin labeled directly with rhodamine (no phalloidin) formed unstable filaments whose average length decreased with time. The number average length of TRITC-phalloidin labeled filaments (Ln) was 4.90 microns, the ratio (rho) of the weight average length to the number average length was 2.06, and the correlation length (1/lambda) was 8.33 microns. These parameters were in good agreement with the values determined by EM for filaments shorter than 8 microns. Passing G-actin through a Sephadex G-150 column before polymerization did not have a significant effect on the distribution of lengths but made filaments more stiff (1/lambda = 12.5 microns). Millimolar concentration of ATP increased the correlation length, and gelsolin had the expected fragmenting effect on filaments. These results show that OM can be used as a fast and reliable method to analyze the distribution and flexibility of actin filaments and suggest that, in spite of extensive manipulation of actin filaments during sample preparation, EM is a valid tool for determination of size parameters of actin filaments.

Actins↗
PubMed1961

Antigenicity of rat collagen. Demonstration of antibody to rat collagen in the renal glomeruli of rats by fluorescence microscopy.

Rabbit serum or globulin, containing antibody to rat collagen, injected intravenously or intracardially into normal or adjuvant-prepared rats becomes fixed in the basement membranes of renal glomeruli and, to a slight extent, of the tubules. When examined by ultraviolet light, this antibody can be identified in tissue sections by the yellow-green fluorescence occurring where the rabbit globulin, associated with the fixed collagen antibody, has reacted with fluorescein-conjugated anti-rabbit globulin from ducks. The reaction of the antibody to rat collagen with its antigen in the kidney is a primary factor in the production of the renal glomerular injury which occurs in rats prepared with adjuvant. Adjacent control sections failed to fluoresce when pretreated with unlabeled anti-rabbit globulin or when treated with heterologous conjugated anti-duck globulin from rabbits. The antibody to rat collagen remains in the kidney as long as 92 days and has been detected as early as 45 minutes after injection. When normal rabbit serum, rabbit anti-fish collagen serum, or rabbit anti-rat collagen serum absorbed with rat collagen was substituted for the rabbit anti-rat collagen serum, fixed antibody could not be demonstrated by fluorescence; but absorption of the anti-rat collagen serum with fish collagen did not affect the antibody fixation. This series of immunologic tests indicates that the anti-collagen serum reacts with its homologous antigen, presumably collagen, in the basement membranes of renal glomeruli and tubules, and that specific antibody can be used to identify collagen in other tissues of the animal body.

Animals↗
PubMedDate not supplied

Subarachnoid haemorrhage in the rat: angiography and fluorescence microscopy of the major cerebral arteries.

A subarachnoid haemorrhage (SAH) in the rat was produced by the injection of blood via a previously implanted catheter connected to the cisterna magna. Repeated angiographical examinations of the vertebro-basilar arteries revealed a biphasic vasospasm with a maximal acute spasm at ten minutes and a maximal late spasm at two days after cisternal blood injection. Fluorescence microscopical examination of the major cerebral arteries at day two after the SAH revealed a reduction in the fluorescence intensity and in the number of histochemically visible sympathetic nerve terminals.

Animals↗
PubMed1983

[Fluorescence microscopy study of fluorescein in the posterior segment of the eye following local application of drugs].

After local application of gentamicin, dexamethasone and tropicamide to rat eyes, no effect on the permeability or the caliber of retinal vessels was observed, nor any diffusion of fluorescein from the choriocapillaris to the outer retinal layers. The qualitative assessment of fluorescence microscopic preparations (and photographic reproductions of them) is relatively unreliable and has led to confusion in the literature. The results presented here, which to some extent contradict earlier findings published in 1971, are discussed. The authors expect to obtain more accurate results from the quantitative method which they are now using.

Animals↗
PubMed1993

Characterization of pigmented granules in minocycline-induced cutaneous pigmentation: observations using fluorescence microscopy and high-performance liquid chromatography.

We describe a technique to facilitate histopathological detection and quantitative measurement of trace amounts of tissue minocycline. In a patient with minocycline-induced hyperpigmentation, a series of biopsies were taken from the affected skin over a period of 54 months, and examined for tissue minocycline deposition. Frozen sections treated with 1 M MgCl2-ethanol yielded minocycline-specific yellowish fluorescence, the location of which corresponded to clumps of granular deposits in the dermis. A decrease in the number of fluorescent granules correlated with the clinical resolution of hyperpigmentation and a significant decrease in the tissue minocycline level. Our data also provide further support for the concept that the pigment granules contain insoluble complexes of minocycline chelated with iron. The fluorescence technique is simple, sensitive, and reproducible.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

Adrenergic innervation of large cerebral blood vessels of the rabbit studied by fluorescence microscopy. Absence of features that might contribute to non-uniform change in cerebral blood flow.

The literature provides evidence for non-uniform regional changes in cerebral blood flow under a variety of circumstances. Possible causes of such changes were sought in the larger cerebral arteries of the rabbit prepared according to the glyoxylic acid fluorescence histochemical technique. The adrenergic innervation of the circle of Willis, the basilar artery and their main branches, although showing small differences, is essentially of uniform density. There is no evidence for collars of adrenergic nerves around the origin of small branches nor for cushions at their orifices. Innervation density appears to diminish as the pial vessels get smaller and all vessels seem to be innervated. Thus non-uniform alterations of cerebral blood flow cannot be accounted for by these factors.

Adrenergic Fibers↗
PubMed2002

Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment.

We compare the axial sectioning capability of multifocal confocal and multifocal multiphoton microscopy in theory and in experiment, with particular emphasis on the background arising from the cross-talk between adjacent imaging channels. We demonstrate that a time-multiplexed non-linear excitation microscope exhibits significantly less background and therefore a superior axial resolution as compared to a multifocal single-photon confocal system. The background becomes irrelevant for thin (< 15 microm) and sparse fluorescent samples, in which case the confocal parallelized system exhibits similar or slightly better sectioning behaviour due to its shorter excitation wavelength. Theoretical and experimental axial responses of practically implemented microscopes are given.

Fluorescence Polarization↗
PubMed2004

Imaging enzyme activity with polarization-sensitive confocal fluorescence microscopy.

We describe a technique for imaging enzyme activity through steady-state fluorescence anisotropy measurements on a per-pixel basis with a confocal microscope. With this method, enzyme activity is reported by changes in the fluorescence anisotropy of a fluorescently labelled substrate. Enzymatic cleavage of the substrate yields smaller labelled fragments that tumble more readily than the intact substrate and therefore yield a lower anisotropy. Anisotropy is recovered to an accuracy of 7% or better on and off the optical axis to depths of 210 microm using objective numerical apertures as high as 0.75. Enzyme imaging experiments were performed with Bodipy-FL-labelled bovine serum albumin (BSA) attached to sepharose beads as a substrate for trypsin and proteinase K. Anisotropy images acquired up to 1 h after enzyme addition revealed more rapid digestion of BSA with proteinase K than with trypsin, but in both cases anisotropy decreased by at least five-fold. Fluorescence lifetime and time-resolved anisotropy decay measurements were made on the construct in fluid solution to reveal the effects of enzyme activity. The Bodipy-FL lifetime increased from 1.34 ns for the construct without enzyme to 5.98 ns after 1 h in the presence of proteinase K. Anisotropy decays yielded average rotational correlation times of 1.13 ns before enzymatic action and 0.27 ns after enzymatic action, consistent with the presence of smaller Bodipy-containing protein fragments. These results suggest wide applicability of the technique in biological systems when used in conjunction with appropriately designed constructs.

Animals↗
PubMed2000

Fluorescein-labeled naloxone binding to mu opioid receptors on live Chinese hamster ovary cells using confocal fluorescent microscopy.

A general method of confocal laser scanning microscopy was used to demonstrate specific binding of fluorescein-labeled naloxone (FNAL, 10-50 nM) to stably transfected mu opioid receptors on live Chinese hamster ovary cells. Nonspecific binding was visually indistinguishable from autofluorescence in cells with intact cell membranes. Fluorescent labeling of cell perimeters, not present in control nontransfected cells, reversed in transfected cells upon washout of FNAL or following the addition of either unlabeled naloxone (25 microM) or the mu specific antagonist CTOP (1 microM). The addition of the delta and kappa specific agonists DPDPE (1 microM) and U50488 (1 microM), respectively, failed to reverse the labeling. Further evidence of specific binding was obtained from kinetic experiments, where it was observed that only transfected cells showed a time-dependent exponential change in fluorescence that permitted estimation of association and dissociation binding rate constants of (5.8+/-0.5, mean+/-S.E.M.)x10(5) M(-1) s(-1) and (3.3+/-0.6)x10(-3) s(-1), respectively and a kinetically derived dissociation constant of 5.7+/-1.4 nM. These estimates were comparable to those obtained under similar conditions in radioligand binding experiments using [3H]-naloxone.

Animals↗
PubMed1983

The development, using poly(Hg-U) in a model system, of a new method to visualize cytochemical hybridization in fluorescence microscopy.

The development, using a model system, of a new method for the detection of cytochemical (in situ) hybrids is described. The method is based on the mercuration of nucleic acids with mercuric acetate. To facilitate hybridization, the acetate ligand is replaced by the CN- ion. In the hybrids formed, the CN- is exchanged for trinitrophenyl(TNP)-glutathione. The TNP-glutathione is subsequently detected by indirect immunofluorescence using anti-TNP antibodies. The feasibility of the approach was investigated using Sepharose- or Sephadex-bound poly(A) and mercurated poly(U). Poly(Hg-U) did hybridize with poly(A)-Sepharose, provided that the acetate ligand was replaced with CN-. The TNP-glutathione hapten thus synthesized bound effectively to mercury-Sepharose but not to amino-Sepharose when the reaction was performed in the dark. Furthermore, binding of TNP-glutathione to Sephadex-bound poly(A) . poly(HG-U) hybrids was detectable with indirect immunofluorescence using anti-TNP antibodies. The fluorescence intensity measured was dependent on the amount of poly(Hg-U) present and on the dilution of the antibody. Nonspecific binding was very low. Calibration of the number of fluorescein molecules found after the complete reaction was performed with fluorescein isothiocyanate-labeled poly(U). It was determined that one fluorochrome molecule per two nucleotides had been obtained, in close agreement with the theoretically expected number. The sensitivity of the method, when applicable to microscopic preparations, is comparable to in situ hybridization with 3H-labeled nucleic acids with a specific activity of 4 x 10(8) dpm/micrograms (two 3H-isotopes per nucleotide) and an exposure time of 1 day. Extension of the method to the cRNA-DNA system and its application to microscopic preparations is under investigation.

Chemical Phenomena↗