SEARCH · Search PubMed

Results for “Fluorescence Microscopy”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

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

PubMed1997

Two-photon fluorescence microscopy of laurdan generalized polarization domains in model and natural membranes.

Two-photon excitation microscopy shows coexisting regions of different generalized polarization (GP) in phospholipid vesicles, in red blood cells, in a renal tubular cell line, and in purified renal brushborder and basolateral membranes labeled with the fluorescent probe laurdan. The GP function measures the relative water content of the membrane. In the present study we discuss images obtained with polarized laser excitation, which selects different molecular orientations of the lipid bilayer corresponding to different spatial regions. The GP distribution in the gel-phase vesicles is relatively narrow, whereas the GP distribution in the liquid-crystalline phase vesicles (DOPC and DLPC) is broad. Analysis of images obtained with polarized excitation of the liquid-crystalline phase vesicles leads to the conclusion that coexisting regions of different GP must have dimensions smaller than the microscope resolution (approximately 200 nm radially and 600 nm axially). Vesicles of an equimolar mixture of DOPC and DPPC show coexisting rigid and fluid domains (high GP and low GP), but the rigid domains, which are preferentially excited by polarized light, have GP values lower than the pure gel-phase domains. Cholesterol strongly modifies the domain morphology. In the presence of 30 mol% cholesterol, the broad GP distribution of the DOPC/DPPC equimolar sample becomes narrower. The sample is still very heterogeneous, as demonstrated by the separations of GP disjoined regions, which are the result of photoselection of regions of different lipid orientation. In intact red blood cells, microscopic regions of different GP can be resolved, whereas in the renal cells GP domains have dimensions smaller than the microscope resolution. Preparations of renal apical brush border membranes and basolateral membranes show well-resolved GP domains, which may result from a different local orientation, or the domains may reflect a real heterogeneity of these membranes.

2-Naphthylamine↗

PubMed1983

[Accumulation of exogenous monoamines in the notochord of lampreys: fluorescence microscopy and radiography study].

Storage of biogenic amines was studied in the notochord of the lamprey (Lampetra planeri) after injection of cold or tritiated amines or their precursors. After injection of dopamine, noradrenaline or 5-hydroxytryptophane, an intense formaldehyde-induced fluorescence was observed in the cytosol of chordal cells and in the perichordal sheat. A similar location occurred after injection of the same tritiated products. No qualitative or significant quantitative differences could be distinguished between the larval and the metamorphosing forms. RO4-4602, a decarboxylase inhibitor, and Nialamide, a monoamine-oxydase inhibitor, did not modify significantly the incorporation of 3H-5-hydroxytryptophan. Using histochemical technique a monoamine oxydase activity could be detected in the nervous system and in the muscle but was not shown in the notochord.

Animals↗

PubMed1980

Fluorescence microscopy of the avian (Gallus domesticus) carotid body.

Sterba's technique to detect neurosecretory substances was applied to carotid body tissue, surrounding branchial derivatives, ganglion nodosum and the carotid arterial wall. Mainly a yellowish-green, a yellowish-red and a bright yellow fluorescence was observed as well as greenish-yellow fibres with varicosities and terminal knobs. The metachromatic reaction of the technique was briefly discussed.

Animals↗

PubMed1984

Rapid detection of fungi in tissues using calcofluor white and fluorescence microscopy.

Rapid intraoperative examination of tissues for fungi is important for the surgical control of infection. Staining of frozen or paraffin-embedded tissues with calcofluor white (CFW) is a rapid nonspecific method for the identification of fungal infection. When viewed with a fluorescence microscope, fungal elements stained with CFW are sharply delineated from surrounding tissue and easily identified. Calcofluor white also stains tissue elements such as keratin, collagen, and elastin, which provide useful landmarks for the examination. To a much lesser degree, bacteria are also stained with CFW.

Animals↗

PubMed2000

Fluorescence microscopy of stimulated Zn(II) release from organotypic cultures of mammalian hippocampus using a carbonic anhydrase-based biosensor system.

We demonstrate here that electrical stimulation of organotypic cultures of rat hippocampus results in the prompt release of significant amounts of Zn(II) by a fluorescence microscopic method. The fluorescence imaging of free Zn(II) is achieved using a highly selective biosensing indicator system consisting of human apo-carbonic anhydrase II (apoCAII) and a fluorescent aryl sulfonamide inhibitor of the enzyme, ABD-N. The apoenzyme and ABD-N in the absence of Zn(II) exhibit weak, reddish fluorescence typical of the ABD-N alone; when Zn(II) is added it binds to the apoenzyme (K(D) = 4 pM), which strongly promotes binding of ABD-N to the holoenzyme (K(D) = 0.9 microM). Binding of ABD-N to the holoenzyme results in a 9-fold increase in apparent quantum yield, significant blue shifts in excitation and emission, an increase in average fluorescence lifetime, a 4-fold increase in the ratio of intensities at 560 and 680 nm, and a large increase in anisotropy. Prior to stimulation, cultures immersed in phosphate-buffered saline with glucose and apoCAII with ABD-N emitted negligible fluorescence, but within 20 s after electrical stimulation a diffuse cloud of greenish fluorescence emerged and subsequently covered most of the culture, indicating release of zinc into the extracellular medium.

Animals↗

PubMed2001

Generalized approach for accelerated maximum likelihood based image restoration applied to three-dimensional fluorescence microscopy.

For deconvolution applications in three-dimensional microscopy we derived and implemented a generic, accelerated maximum likelihood image restoration algorithm. A conjugate gradient iteration scheme was used considering either Gaussian or Poisson noise models. Poisson models are better suited to low intensity fluorescent image data; typically, they show smaller restoration errors and smoother results. For the regularization, we modified the standard Tikhonov method. However, the generic design of the algorithm allows for more regularization approaches. The Hessian matrix of the restoration functional was used to determine the step size. We compared restoration error and convergence behaviour between the classical line-search and the Hessian matrix method. Under typical working conditions, the restoration error did not increase over that of the line-search and the speed of convergence did not significantly decrease allowing for a twofold increase in processing speed. To determine the regularization parameter, we modified the generalized cross-validation method. Tests that were done on both simulated and experimental fluorescence wide-field data show reliable results.

Algorithms↗

PubMed1980

Fluorescent microscopy of glycol methacrylate-embedded tissue.

Formalin fixed, glycol methacrylate-embedded tissue samples can easily be used for immunofluorescent studies by direct and indirect fluorescein isothiocyanate staining after treatment with protease V to unmask antigenicity. It is, therefore, possible to use serial 2-micron glycol methacrylate-embedded sections for immunologic studies, as well as conventional and special stains.

Acrylic Resins↗

PubMed1993

Evidence from fluorescence microscopy and comparative studies that rat, ovine and bovine colonic crypts are absorptive.

1. To test whether colonic crypts are secretory or absorptive interstitial [Na+] in rat descending colonic mucosa is determined using video-enhanced imaging of the impermeant acid form of the fluorescent Na+ probe SBFI (Molecular Probes) and intracellular [Na+] is monitored with SBFI (AM form). In rat descending colonic mucosa perifused with isotonic Tyrode solution interstitial [Na+] = 500-650 mM. Following exposure to Tyrode solution containing theophylline (10 mM) interstitial [Na+] falls by 300-450 mM within 1 min. Exposure to amiloride (0.2 mM) reduces the intracellular [Na+] from ca 25 to 12 mM within 15 min and concurrently decreases [Na+] in the interstitial fluid surrounding the crypts at the mucosal surface by approximately 200 mM. 2. The route of fluid inflow across the rat colonic mucosa is directly traced by perifusing with Tyrode solution containing the impermeant fluorescent dye, fluorescein disulphonate (FS). FS accumulates rapidly within crypt lumens of control tissues to a 2-fold higher concentration than in the external bathing solution, but FS does not accumulate in crypts of tissues treated with azide (2 mM). The increment in FS accumulation within the crypt lumen above the bulk solution decreases by 80% within 1 min following exposure to theophylline (10 mM), indicating that fluid absorption into crypts is reduced. Estimates of the total fluid influx from the rate and extent of FS concentration polarization within crypts indicate that it is sufficient to account for the entire transcolonic fluid absorption. 3. Comparative studies of isolated bovine and ovine colon were also undertaken to investigate the failure of bovine colon to generate a hypertonic absorbate and hence its incapacity to produce hard faeces. The interstitial fluid surrounding ovine colonic crypts is hypertonic to the bulk solution, whereas the interstitial fluid surrounding bovine colonic crypts is nearly isotonic with the bathing solution. Additionally, fluorescein disulphonate accumulates within ovine colonic crypt lumens by concentration polarization, whereas no concentration of FS occurs within bovine colonic crypt lumens. This corroborates the view that a hypertonic interstitial fluid is absent from bovine colon mainly because of a high rate of transepithelial leakage of low molecular weight solutes via paracellular routes.

Animals↗

PubMed2006

Visualizing calcium signaling in cells by digitized wide-field and confocal fluorescent microscopy.

Calcium (Ca2+) is a fundamentally important component of cellular signal transduction. Dynamic changes in the concentration of Ca2+ ([Ca2+]) in the cytoplasm and within organelles are tightly controlled and regulate a diverse array of biological activities, including fertilization, cell division, gene expression, cellular metabolism, protein biosynthesis, secretion, muscle contraction, intercellular communication, and cell death. Measurement of intracellular [Ca2+] is essential to understanding the role of Ca2+ and for defining the underlying regulatory mechanisms in any cellular process. A broad range of synthetic and biosynthetic fluorescent Ca2+ sensors are available that enable the visualization and quantification of subcellular spatio-temporal [Ca2+] gradients. This chapter describes the application of wide-field digitized video fluorescence microfluorometry and confocal microscopy to quantitatively image Ca2+ in cells with high temporal and spatial resolution.

Animals↗

PubMed1995

Location by fluorescence microscopy of glycosidases and a xylanase in the anaerobic gut fungi Caecomyces communis, Neocallimastix frontalis, and Piromyces rhizinflata.

beta-D-Glucosidase, beta-D-fucosidase, beta-D-xylosidase, and beta-D-cellobiopyranosidase activities in Caecomyces communis, Neocallimastix frontalis, and Piromyces rhizinflata, located with fluorescent conjugates, occur throughout the whole thallus as from zoospore germination and disappear before sporulation. beta-D-Galactosidase and alpha-L-arabinopyranosidase activities are low or nonexistent. A xylanase, detected by indirect immunofluorescence, was observed at the surface of the vegetative cells, vesicles, or rhizoids. Cross-reactions prove the existence of analogies in structure among the enzymes of these anaerobic gut fungi.

Animals↗

PubMed2003

Comparison of reagents for shape analysis of fixed cells by automated fluorescence microscopy.

BACKGROUND: Cell size and shape have been implicated as potentiators of intracellular signaling events and as indicators of abnormal cell behavior. Automated microscopy and image analysis can provide quantitative information about the size and shape of cultured cells, but it requires that the edge of a cell be clearly identified. Generating adequate contrast at the edge of thin well-spread cells can be challenging. METHODS: We compared six (five chemically reactive and one lipophilic) fluorescent molecules--5-chloromethyl fluorescein diacetate (CMFDA, CellTracker green), fluorescein-5-maleimide, fluorescein-5-isothiocyanate (FITC), 5-iodoacetamidofluorescein, 5(6)-carboxy fluorescein-N-hydroxysuccinimidyl ester, and N-fluorescein-1,2-dihexadecanoyl-sn-glycerol-3-phosphoethanolamine--for their effectiveness as stains for automated morphology analysis of fixed cells. RESULTS: Formaldehyde-fixed rat aortic smooth muscle cells stained with fluorescein-5-maleimide or FITC exhibited an average intensity that was at least twofold greater than cells stained with CMFDA even when subjected to a 25-fold shorter exposure time. Cell area determined with the higher intensity stains was less sensitive to threshold settings during automated cell morphology analysis. CONCLUSION: A procedure that includes the use of fluorescein-5-maleimide or FITC for staining fixed cell provides sensitivity sufficient to permit rapid, automated, morphologic analysis of well-spread fixed cells.

Animals↗

PubMed1999

A quick molecular method for the simultaneous detection in spermatozoa of nuclear, acrosomal and axonemal structure by fluorescent microscopy.

Ejaculated spermatozoa from infertile men presenting to our laboratory for semen analysis were processed with a new molecular method which reveals simultaneously, in the same sperm cell, the status of the acrosome, by testing the hyaluronidase content, the texture of the nucleus, by checking the DNA strands breaks, and the structure of the axoneme, revealing the tubulin content. The presence of hyaluronidase and tubulin is essential for the sperm function, and the analysis of the DNA status reveals the eventual apoptotic process. Using this method in normal spermatozoa, the staining of the acrosomal hyaluronidase reveals, by yellow-green fluorescence, the shape of the acrosomal complex and its texture. At the same time, in the same sperm cell, the staining of the axonemal tubulin demonstrates, by a red labeling, the presence of the protein and therefore the consistence of the axonemal structure. Simultaneously, at the head level, the absence of red labeling from nuclear DNA indicates that the apoptotic process is not present. This protocol allows quantification of the frequency of the presence of normal or abnormal spermatozoa, by an easy scoring and calculation of the apoptotic sperm or of the sperm with generic defects at acrosomal or flagellar level. The percentage of normal spermatozoa evaluated by the triple staining method has been compared with the results of the PAP staining and of the ultrastructural analysis, statistically elaborated. Triple staining results more severe than the PAP method, but TEM analysis is the finest technique to detect sperm abnormality because it considers the entire panel of sperm defects.

Acrosome↗

PubMed1980

Visualization of specific angiotensin II binding sites in the brain by fluorescent microscopy.

The organum vasculosum of the lamina terminalis has been implicated as the site of receptors mediating central responses of angiotensin II. Up to now, this had been based on indirect evidence, but direct visualization of angiotensin II at its site of action has now been achieved by the use of a biologically active fluorescent angiotensin II agonist. The ventricular surface of the organum vasculosum lamina terminalis showed intense fluorescence, which was virtually eliminated by an excess of unlabeled angiotensin II.

Angiotensin II↗

PubMed1993

Flow injection fluorescence microscopy: a novel tool for the study of cells through controlled perfusion.

Current methods of microscope stage perfusion do not take full advantage of existing technology for precise fluid control. The concept of flow injection, used extensively by analytical chemists, is described and its application to the fluorescence microscopic study of cultured cells is proposed. Using this technique, cells may be exposed to single or multiple reagent zones of almost any profile, sequence, and duration, with computer-controlled precision. A flow injection system is employed in conjunction with a novel perfusion chamber--the fountain cell. The ability of the flow injection system to perfuse cells with a reagent with a reproducibility of 1% RSD is demonstrated. The system was used to monitor changes in calcium levels in baby hamster kidney cells loaded with FURA-2 as a result of stimulation with a precisely timed concentration of ionomycin. The unique feature of the technique is that it allows a series of responses of a given cell to be directly compared to each other.

Animals↗

PubMed2004

Tip-enhanced fluorescence microscopy at 10 nanometer resolution.

We demonstrate unambiguously that the field enhancement near the apex of a laser-illuminated silicon tip decays according to a power law that is moderated by a single parameter characterizing the tip sharpness. Oscillating the probe in intermittent contact with a semiconductor nanocrystal strongly modulates the fluorescence excitation rate, providing robust optical contrast and enabling excellent background rejection. Laterally encoded demodulation yields images with <10 nm spatial resolution, consistent with independent measurements of tip sharpness.

Lasers↗

PubMed1991

Intralobular heterogeneity of carbon tetrachloride-induced oxidative stress in perfused rat liver visualized by digital imaging fluorescence microscopy.

Spatial and temporal alterations in carbon tetrachloride-induced oxidative stress were studied in perfused hepatic microcirculatory units of the rat by digital imaging microscopic fluorography using dichlorofluorescin diacetate, a hydroperoxide-sensitive fluorogenic probe. The surface of the liver loaded with dichlorofluorescin was microscopically visualized, and the fluorescence of dichlorofluorescin (DCF), a highly fluorescent molecule generated by hydroperoxide-mediated oxidation of dichlorofluorescin, was digitally processed. After completing the experiments, fluorescein-labeled albumin was injected into the perfusate to confirm the state of sinusoidal perfusion and the topography of hepatic microangioarchitecture in the area studied. During transportal infusion of carbon tetrachloride, DCF fluorescence was observed predominantly in the pericentral area and was attenuated by pretreatment with SKF-525A, suggesting the involvement of cytochrome P-450. After peaking at the maximum level, the pericentral DCF fluorescence gradually decreased in parallel to the loss of viability, implicating the causal role of intracellular hydroperoxide formation in hepatocellular injury. In retrogradely perfused liver, in which intralobular O2 gradient was reversed, no significant activation of DCF fluorescence was observed among hepatic lobules. It is therefore conceivable that the zonal heterogeneity of carbon tetrachloride-induced lipid peroxidation may depend on at least two factors, sinusoidal oxygenation and the intralobular distribution of cytochrome P-450. Furthermore, development of the current technique will provide a useful method to investigate the microtopography of oxidative stress in organ microcirculatory units.

Animals↗

PubMed2005

Fluorescence microscopy for the evaluation of the margins of Class V restorations in vitro.

PURPOSE: First, to elucidate the cause of the appearance of "white lines" in Class V restorations as seen under the light microscope and their enhancement when using a fluorescent microscope (FM); second, to compare the results of the FM quantitative marginal analysis to those of the SEM quantitative marginal analysis. MATERIALS AND METHODS: Thirty-two standardized Class V fillings, with half of the preparation in dentin and half in enamel, were placed in 16 human caries-free premolars on the buccal and lingual surface using 4 adhesive systems (Clearfil SE, Syntac, 2 experimental self-etching adhesives) and the composite Tetric Ceram. All teeth were connected to a device containing horse serum to simulate dentinal fluid and subjected to thermomechanical loading (1,200,000 cycles at 49 N, 3000 cycles at 5 degrees C/55 degrees C). The restoration margins of 24 fillings were directly evaluated with FM--after the fabrication of replicas--and with SEM. Eight fillings were used to elucidate the cause of the fluorescent signal, by examining them with FM and different filters under dry and moist conditions, and also with dark field microscopy and CLSM. Further, two Class V fillings with the same composite but without the fluorescent substances were made and evaluated. Selected specimens were cut in the bucco-oral direction to analyze the restorative interface on section replicas with SEM. To detect differences between FM and SEM, the Wilcoxon test was performed (p < 0.05). To evaluate the degree of agreement between the two evaluation methods, a linear regression analysis was performed and the Spearman correlation coeffcient was calculated. RESULTS: The evaluation of Class V restorative margins by SEM, dark field microscopy, and CLSM revealed that the phenomenon seems to be an optical effect caused by the debonding of the composite from the underlying substrate creating another refractive environment when air penetrates into the gap. The effect depends on the geometry of the cavity design, the absence of moisture, and the use of a fluorescent composite material. The light source that produced the best visible results covered both ultraviolet and the blue part of white light (350 to 460 nm), producing light green lines and green areas. Those green or white areas as seen with the light microscope were related to gaps in the interface between tooth substance and composite. With the exception of one of the experimental self-etching adhesives, the mean percentage of continuous margin of the 4 groups showed no statistically signifcant dfference for the two test methods. Linear regression analysis revealed a good correlation between the two test methods for dentin margins and a moderate correlation for enamel margins. CONCLUSION: Fluorescence microscopy seems to be a useful tool for evaluating dentinal and enamel margins of Class V restorations in vitro.

Adhesives↗

PubMed2000

Two-photon fluorescence microscopy studies of bipolar tetraether giant liposomes from thermoacidophilic archaebacteria Sulfolobus acidocaldarius.

The effects of temperature and pH on Laurdan (6-lauroyl-2-(dimethylamino)naphthalene) fluorescence intensity images of giant unilamellar vesicles (GUVs) ( approximately 20-150 microm in diameter) composed of the polar lipid fraction E (PLFE) from the thermoacidophilic archaebacteria Sulfolobus acidocaldarius have been studied using two-photon excitation. PLFE GUVs made by the electroformation method were stable and well suited for microscopy studies. The generalized polarization (GP) of Laurdan fluorescence in the center cross section of the vesicles has been determined as a function of temperature at pH 7.23 and pH 2.68. At all of the temperatures and pHs examined, the GP values are low (below or close to 0), and the GP histograms show a broad distribution width (> 0.3). When excited with light polarized in the y direction, Laurdan fluorescence in the center cross section of the PLFE GUVs exhibits a photoselection effect showing much higher intensities in the x direction of the vesicles, a result opposite that previously obtained on monopolar diester phospholipids. This result indicates that the chromophore of Laurdan in PLFE GUVs is aligned parallel to the membrane surface. The x direction photoselection effect and the low GP values lead us to further propose that the Laurdan chromophore resides in the polar headgroup region of the PLFE liposomes, while the lauroyl tail inserts into the hydrocarbon core of the membrane. This unusual L-shaped disposition is presumably caused by the unique lipid structures and by the rigid and tight membrane packing in PLFE liposomes. The GP exhibited, at both pH values, a small but abrupt decrease near 50 degrees C, suggesting a conformational change in the polar headgroups of PLFE. This transition temperature fully agrees with the d-spacing data recently measured by small-angle x-ray diffraction and with the pyrene-labeled phosphatidylcholine and perylene fluorescence data previously obtained from PLFE multilamellar vesicles. Interestingly, the two-photon Laurdan fluorescence images showed snowflake-like lipid domains in PLFE GUVs at pH 7.23 and low temperatures (<20 degrees C in the cooling scan and <24 degrees C in the heating scan). These domains, attributable to lipid lateral separation, were stable and laterally immobile at low temperatures (<23 degrees C), again suggesting tight membrane packing in the PLFE GUVs.

2-Naphthylamine↗