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

Computerised image analysis in conjunction with fluorescence microscopy for the study of blood-brain barrier permeability in vivo.

The present paper describes a new method using computerised image analysis techniques for quantification of tracer extravasation over the blood-brain barrier as studied by intravital fluorescence microscopy. Cats were equipped with an open cranial window and continuously infused with fluorescein isothiocyanate-labelled dextran (FITC-dextran, mol. wt. 70,000) to maintain a steady plasma concentration. Several cortical fields were recorded in each experiment and the images stored on video tape for off-line analysis. This procedure, which largely eliminates the superficial pial vasculature and allows extraction of the extravasation areas, consists of the following steps: (1) averaging of images, (2) software shading correction based on the original images for compensation of optical non-uniformity, (3) correction of displacement artefacts, (4) intensity adjustment, (5) generation of subtraction images by subtracting the first image of a series from the subsequent ones, (6) median filtering and thresholding, (7) a length recognition algorithm, and (8) elimination of small areas. Compared to the previously described method, step (2) has been newly developed and steps (4) and (8) added to enhance sensitivity for detecting tracer extravasation. The degree of extravasation in a cortical field at a given time point [E(f) value] was calculated as the mean intensity of the remaining pixels. The E(f) is a quantitative value computed by a fully automatised procedure which takes into account the number, as well as the size and intensity, of extravasation areas in a given cortical field. The E(f) values obtained at different times in a series of experiments were averaged to give the E(I) value.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed2004

Fingerprints' third dimension: the depth and shape of fingerprints penetration into paper--cross section examination by fluorescence microscopy.

In an attempt to maximize the yield of latent fingerprints from paper items, we conducted a study of a fundamental process between fingerprint deposits and paper. Fingerprint ridges have been observed in the cross section of paper by fluorescence microscopy. It was possible to see, for the first time, how residue from fingerprint ridges is embedded in paper. Undeveloped, latent fingerprints, as well as latent prints developed by the two fluorogenic reagents, DFO and 1,2-indanedione, have been examined. The shape and depth of penetration of fingerprints vary with different types of paper. An inverse relationship between the smoothness of the paper and the penetration depth was observed: higher smoothness values result in lower depths of penetration. High quality prints appear to correlate with an optimal penetration depth-between 40 and 60 microns.

Aza Compounds↗
PubMed1990

[The evaluation of fertility characteristics in hens using fluorescence microscopy].

A method for the assessment of fertility in domestic hens is described. Vitelline membranes of fertilized eggs were coloured with fluorochrome DAPI, following which the DNA of sperms on these membranes has been examined with the help of fluorescence microscopy. After insemination with frozen semen there exist significant correlations between the number of sperms on the vitelline membranes on the one hand and 1) fertilization rates; 2) the length of fertile periods. Furthermore, it was observed that with higher numbers of sperms on the membranes, the length of the fertile periods tends to be shorter than with smaller numbers of sperm.

Animals↗
PubMed2002

Total internal reflection fluorescence microscopy for single-molecule imaging in living cells.

Marvelous background rejection in total internal reflection fluorescence microscopy (TIR-FM) has made it possible to visualize single-fluorophores in living cells. Cell signaling proteins including peptide hormones, membrane receptors, small G proteins, cytoplasmic kinases as well as small signaling compounds have been conjugated with single chemical fluorophore or tagged with green fluorescent proteins and visualized in living cells. In this review, the reasons why single-molecule analysis is essential for studies of intracellular protein systems such as cell signaling system are discussed, the instrumentation of TIR-FM for single-molecule imaging in living cells is explained, and how single molecule visualization has been used in cell biology is illustrated by way of two examples: signaling of epidermal growth factor in mammalian cells and chemotaxis of Dictyostelium amoeba along a cAMP gradient. Single-molecule analysis is an ideal method to quantify the parameters of reaction dynamics and kinetics of unitary processes within intracellular protein systems. Knowledge of these parameters is crucial for the understanding of the molecular mechanisms underlying intracellular events, thus single-molecule imaging in living cells will be one of the major technologies in cellular nanobiology.

Animals↗
PubMed1987

Na-H and Cl-HCO3 exchange in rabbit oxyntic cells using fluorescence microscopy.

We have used the fluorescence intensity ratio (excitation 490/439; emission 520-550 nm) of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to measure intracellular pH (pHi) in single oxyntic cells (OCs) within intact gastric glands isolated from rabbits. The fluorescence ratio was converted to pHi by exposing cells to high [K] Ringer plus 10(-5) M nigericin. Ratios varied linearly with pHi over the physiological range. When the bathing solution was changed from NaCl to Na gluconate Ringer, pHi increased from 7.0 to 7.4. The increase of pHi occurred equally rapidly in nominally CO2-HCO3-free solutions and in solutions containing 5% CO2 and 25 mM HCO3. This effect was reversible and blocked by 2 X 10(-4) M 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). The DIDS block was bypassed by 10(-5) M tri-n-butyltin, a pharmacological Cl-OH exchanger. When the bathing solution was changed from NaCl to N-methyl-D-glucamine (NMG) Cl Ringer, pHi decreased reversibly from 7.0 to 6.8. It was also found that changing from NMG gluconate to Na gluconate Ringer caused pHi to increase from 7.1 to 7.3, and this alkalinization was blocked by 10(-3) M amiloride; changing from NMG gluconate to NMG Cl Ringer caused pHi to decrease to 6.7. We conclude that OCs contain separate Na-H and Cl-HCO3 exchangers. Similarities and differences between these exchangers and those present in other cell types are discussed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

Laser-induced fluorescence microscopy of normal colon and dysplasia in colonic adenomas: implications for spectroscopic diagnosis.

OBJECTIVES: To determine what structures fluoresce and to what extent in normal colon and colonic adenomas to fully exploit laser-induced fluorescence spectroscopy as a tool for the diagnosis of dysplasia at endoscopy. METHODS: Unstained frozen sections of normal colon and colonic adenomas were studied by fluorescence microscopy under 351-364-nm argon ion laser excitation. Tissue fluorescence was observed and compared to morphology in serial sections stained with hematoxylin and eosin (H&E), Movat pentachrome, mucicarmine, and oil red O. RESULTS: In normal colon, fluorescence correlated morphologically with connective tissue fibers (principally collagen) in all layers of the bowel wall and with cytoplasmic granules within eosinophils present between the crypts in the lamina propria of the mucosa. Fluorescence of absorptive cells in normal crypts was very faint, and Goblet cells did not fluoresce. However, marked fluorescence was observed in the cytoplasms of dysplastic epithelial cells in the crypts of colonic adenomas. Fewer fluorescent connective tissue fibers were present in the lamina propria of colonic adenomas resulting in decreased fluorescence intensity as compared to that of normal colon. Fluorescent eosinophil granules were present in larger numbers in adenomas as compared with normal colon. CONCLUSION: Laser-induced fluorescence in normal colon and colonic adenomas correlates with morphology. Previous reported differences in laser-induced fluorescence emission spectra of normal colon and colonic adenomas obtained in vitro and in vivo may be due to differences in the cytoplasmic fluorescence between the dysplastic epithelium in colonic adenomas and normal colonic epithelium. Laser-induced fluorescence spectroscopy may be useful in studying other forms of epithelial dysplasia such as that which occurs in ulcerative colitis.

Adenoma↗
PubMed1992

Total internal reflection fluorescence microscopy (TIRFM). II. Topographical mapping of relative cell/substratum separation distances.

A simplified model of TIRF optics was used to quantitate the relative membrane/substratum separation distances from the spatial pattern of TIRF image brightness. Phase-contrast and total internal reflection fluorescence microscopy (TIRFM) images were collected of bovine aortic endothelial cells (BAEC) plated onto glass microscope slides for 15 min, 30 min and 24 h. BAEC adherent for 15 min showed an absence of a focal contact morphology, with the region of closest apposition beneath the cell center. After 30 min, multiple contacts with the surface were established and the morphology became more irregular. BAEC attached for 24 h showed well-defined focal contact regions aligned in characteristically striated patterns. The relative distance between closest and farthest membrane/substratum separations are consistent with reported distance between focal and matrix contacts. Topographical maps of membrane/substratum separation distances over the entire ventral surface of the plated cells were constructed to demonstrate the utility of quantitative TIRF microscopy.

Animals↗
PubMed2003

A comprehensive comparison of Ziehl-Neelsen and fluorescence microscopy for the diagnosis of tuberculosis in a resource-poor urban setting.

SETTING: Nairobi City Council Chest Clinic, Kenya. OBJECTIVES: To establish the efficiency, costs and cost-effectiveness of six diagnostic strategies using Ziehl-Neelsen (ZN) and fluorescence microscopy (FM). DESIGN: A cross-sectional study of 1398 TB suspects attending a specialised chest clinic in Nairobi subjected to three sputum examinations by ZN and FM. Lowenstein-Jensen culture was used as the gold standard. Cost analysis included health service and patient costs. RESULTS: Of 1398 suspects enrolled, 993 (71%) had a complete diagnostic work-up involving three sputum specimens for ZN and FM, culture and chest X-ray (CXR). Irrespective of whether ZN or FM was used on one, two or three smears, the overall diagnostic process detected 92% culture-positive cases. Different strategies affected the ratio of smear-positive to smear-negative TB; however, FM was more sensitive than ZN (P < 0.001). FM performance was not affected by the patient's HIV status. The cost per correctly diagnosed smear-positive case, including savings, was 40.30 US dollars for FM on two specimens compared to 57.70 US dollars for ZN on three specimens. CONCLUSION: The FM method used on one or two specimens is more cost-effective and shortens the diagnostic process. Consequently, more patients can be put on a regimen for smear-positive TB, contributing to improved treatment and reducing transmission.

Adolescent↗
PubMed2003

Cleavage of stretched DNA on glass by histones observed with fluorescence microscopy.

A stretched single lambda DNA molecule labeled with TOTO-1 dye molecules was immobilized on a cover slip by casting a solution of DNA-lipid polyion complex containing TOTO-1 dye, and were loaded with an aqueous solution of histone. Fluorescence microscopy applied to the single DNA showed that (1) the TOTO-1 dye molecules intercalated in the stretched DNA molecule were vertically aligned almost completely with respect to the DNA axis, and (2) the stretched DNA was cleaved by interaction with histone molecules.

DNA↗
PubMed1977

Glomerulopathy and arteriolopathy in congenital nephrotic syndrome: light, electron, and fluorescence microscopy studies.

This report describes the light, electron, and fluorescence microscopy studies of the kidney from a one month old, prematurely born infant with the nephrotic syndrome. Light microscopy revealed enlargement of Bowman's space, an excessive number of glomerular epithelial cell nuclei, and cystic dilatation of cortical tubules. By electron microscopy two disparate types of glomerular lesions were found: a thin basement membrane with fusion of foot processes suggestive of lipoid nephrosis and linear electron-density in the basement membrane with excessive mesangial matrix and capillary collapse suggesting an antibasement membrane type of glomerulonephritis or mesangial sclerosis. Conspicuous cystic spaces were found in the proximal tubules only. In the arterioles there were cystic spaces in the endothelial cells and smooth muscle cells, thickening of the basement membranes of endothelial cells and smooth muscle cells, and excessive amounts of nonstriated fibrils in the subendothelial basement membrane. Masses of fibrin were noted in the lumina of veins, peritubular capillaries, and interstitium between veins and peritubular capillaries. Such multiple glomerular and arteriolar lesions have not been reported previously in congenital nephrotic syndrome. These lesions suggest an antigen-antibody reaction in the mother that might have been directed toward the glomerular basement membrane in the fetus, producing these lesions.

Biopsy↗
PubMed1992

Epi-fluorescence microscopy and image analysis used to measure diffusion coefficients in gel systems.

A method using epi-fluorescence microscopy and image analysis has been developed to follow and quantify the diffusion of fluorescent compounds through gels. Two mathematical approaches were employed to calculate diffusion coefficients. The spatial resolution provided by the fluorescence microscope allowed diffusion to be followed over very short distances; accordingly diffusion coefficients were obtained within minutes, even for slowly diffusing systems. The method was successfully applied to the diffusion of macromolecules into agar, carbopol and mucus gel systems.

Algorithms↗
PubMed2005

Docking and fusion of insulin secretory granules in SUR1 knock out mouse beta-cells observed by total internal reflection fluorescence microscopy.

To explore how the sulfonylurea receptor (SUR1) is involved in docking and fusion of insulin granules, dynamic motion of single insulin secretory granules near the plasma membrane was examined in SUR1 knock-out (Sur1KO) beta-cells by total internal reflection fluorescence microscopy. Sur1KO beta-cells exhibited a marked reduction in the number of fusion events from previously docked granules. However, the number of docked granules declined during stimulation as a consequence of the release of docked granules into the cytoplasm vs. fusion with the plasma membrane. Thus, the impaired docking and fusion results in decreased insulin exocytosis from Sur1KO beta-cells.

ATP-Binding Cassette Transporters↗
PubMed1992

Distribution of F-actin and fodrin in the hair cells of the guinea pig cochlea as revealed by confocal fluorescence microscopy.

We double-stained paraformaldehyde fixed guinea pig cochleas with rhodaminated phalloidin to detect F-actin and with a monoclonal antibody against non-erythroid spectrin (fodrin). The hair cells were studied in surface specimens of the organ of Corti with confocal fluorescence microscopy. In serial optical sections, phalloidin stained the stereocilia, cuticular plate, and a circumferential ring beneath it in the inner and outer hair cells (IHCs and OHCs). The cytoplasm of the IHCs and the OHCs was unlabelled, but the infracuticular network of the OHCs in the upper turns showed a strong reaction. The lateral plasma membrane was unreactive with phalloidin in the IHCs and OHCs, except in the basal turn, where a moderate reaction, probably representing actin of Deiter's cups, was seen along the lateral walls of the basal pole of the OHCs. Fodrin was similarly seen in the cuticular plate, in a circumferential ring beneath it, and in the infracuticular network of the apical OHCs. The most interesting finding was the fodrin-specific distinct labelling of the lateral cell surface in the OHCs of the basal cochlear turn. This staining diminished towards the apex and was practically absent in the OHCs located above the level of 15 mm from the round window. The lateral cell surface of IHCs showed moderate fodrin labelling in all cochlear turns. This staining was much weaker than that seen in the basal OHCs. Fodrin labelling revealed deformation from the regular cylindrical shape in midportion of the OHC bodies in the basal turn of the cochlea.

Actins↗
PubMed1998

Uncontrolled diabetes hinders bone formation around titanium implants in rat tibiae. A light and fluorescence microscopy, and image processing study.

This study examined the influence of diabetes mellitus on bone formation around cylindrical titanium (Ti) implants (1.0 mm in diameter and 1.5 mm in length) inserted transcortically and extending into the medullary canal of rat tibiae using light and fluorescence microscopies and image processing. Forty-eight male Wistar King A rats (age 5 weeks) were used in this experiment. Streptozotocin was injected intraperitoneally to induce diabetes and the serum glucose concentration was checked to ensure the induction of diabetes prior to implant placement and at the time of sacrifice. The animals were sacrificed 7, 28, 56, or 84 days after placement. Toluidine blue-stained undecalcified sections were prepared for histological observation and image analysis. The Ti implants in the control group became increasingly encapsulated with a bone layer. The implants in the diabetes-induced (DI) group were also surrounded with a thin bone layer. Abundant adipocytes were observed in the DI group as compared with the control group. Quantitative evaluation indicated that the control group showed a significantly higher percent of bone contact, and thickness of surrounding bone and area than the DI group. Consequently, the present study suggests that uncontrolled diabetes would hinder bone formation around Ti implants in rats.

Adipocytes↗
PubMed1982

Digitized video fluorescence microscopy studies of adriamycin interaction with single P388 leukemic cells.

We have evaluated a new fluorescent method, the digitized video fluorescence microscopy technique, for the analysis of Adriamycin drug levels in single-cell suspension. This method uses a Leitz microscope equipped with an HBO 50 watt mercury source; the vertical body of the microscope is attached to an intensified silicone intensifier video camera with its output coupled to a video cassette recorder and to an Apple II microcomputer equipped with a video image digitizer. Using this technique, we were able to corroborate previous findings of decreased uptake and increased efflux in resistant as compared to sensitive P388 leukemic cells. This instrument may have wide applications in the study of anthracycline cell interaction or of any other drug with fluorescent properties.

Adsorption↗
PubMed1989

Measurement of volume injected into individual cells by quantitative fluorescence microscopy.

Pressure microinjection is frequently used to introduce substances into mammalian cells, but precise quantitation of the volume injected into individual cells has been difficult. A simple and reliable procedure for determining the volume injected was developed in order to determine what intracellular concentration of AMP-PNP was necessary to inhibit specific cellular processes. The technique uses fluorescent Lucifer Yellow-labeled dextrans in the microinjection buffer and quantitative fluorescence microscopy to measure the fluorescence intensity of the injected cell. The volume injected is computed from a standard curve derived from the volume and fluorescence of spherical, microscopic droplets of Lucifer Yellow dextran solution. The droplets are ejected from a micropipet into immersion oil where they sink to rest on a siliconized coverslip. For the measurement of fluorescence, an inexpensive photomultiplier system that is attached to a fluorescence microscope is described. The potential uses of this method for other microassays are discussed.

Adenylyl Imidodiphosphate↗
PubMed1982

Time-resolved fluorescence microscopy of hematoporphyrin-derivative in cells.

This work presents measurements of time-resolved fluorescence microscopy of hematoporphyrin-derivative (HpD) in single cells of mice tissue (both tumor and normal cells), in HeLa Cells, and in solution. The measurements were performed using a pulsed-laser microfluorometer with high spatial and temporal resolution. In agreement with the results obtained with other techniques, it has been found that the tumor cells examined present an HpD uptake about five times higher than that of the normal cells of the corresponding tissue and that, within a cell, HpD become localized mainly in the cytoplasm. It has also been found that the fluorescence decay time is different in cells as compared with solution, and that the presence of HpD stabilizes cell auto-fluorescence. These results are discussed.

Animals↗
PubMed1997

Fluorescence microscopy and three-dimensional imaging of the porcine corneal keratocyte network.

BACKGROUND: Little is known about the spatial arrangement and the corresponding morphometric data describing the living keratocyte network. For determination of alterations in corneal diseases it is crucial to know the morphology of the keratocyte network in the healthy state. Porcine cornea was used as a model tissue because it allows the study of species differences. METHODS: Corneas from freshly enucleated pig eyes were stained with calcein AM and ethidium homodimer and examined by confocal laser scanning microscopy. High-resolution fluorescence images were used for three-dimensional reconstructions from which cell density and volume density were determined by computer-aided morphometry. RESULTS: Three keratocyte subpopulations were distinguished and visualized in their spatial arrangement. Significant differences with respect to both shape and fluorescence intensity distribution of the cell bodies were found. Cell volume density was 7.7% in the anterior stroma, 13.7% in the central stroma and 11.8% in the posterior stroma. CONCLUSION: The technique described allowed good visualization of the spatial arrangement of the keratocyte network. Combined with morphometric methods, the analysis of the state of the cornea yields a quantitative description. The method is expected to be useful for the determination of morphological alterations in corneal disease or following surgical treatment.

Animals↗