Search PubMed⌕ Search

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.

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

At least 73 records · Page 4Linked to original sources
PubMed2004

Robust incremental compensation of the light attenuation with depth in 3D fluorescence microscopy.

Summary Fluorescent signal intensities from confocal laser scanning microscopes (CLSM) suffer from several distortions inherent to the method. Namely, layers which lie deeper within the specimen are relatively dark due to absorption and scattering of both excitation and fluorescent light, photobleaching and/or other factors. Because of these effects, a quantitative analysis of images is not always possible without correction. Under certain assumptions, the decay of intensities can be estimated and used for a partial depth intensity correction. In this paper we propose an original robust incremental method for compensating the attenuation of intensity signals. Most previous correction methods are more or less empirical and based on fitting a decreasing parametric function to the section mean intensity curve computed by summing all pixel values in each section. The fitted curve is then used for the calculation of correction factors for each section and a new compensated sections series is computed. However, these methods do not perfectly correct the images. Hence, the algorithm we propose for the automatic correction of intensities relies on robust estimation, which automatically ignores pixels where measurements deviate from the decay model. It is based on techniques adopted from the computer vision literature for image motion estimation. The resulting algorithm is used to correct volumes acquired in CLSM. An implementation of such a restoration filter is discussed and examples of successful restorations are given.

Algorithms↗
PubMed1987

[Stromal tumor of the testis with Leydig cell components. Light and electron microscopy analysis and fluorescence microscopy demonstration of F-actin].

A testicular gonadal stromal tumor with Leydig cells and mesenchymal cells resembling to Sertoli cells is presented. At the ultrastructural level most of the tumor cells could only be classified as endocrine differentiated cells. Because of their uncharacteristic organelles no Sertoli cells could be demonstrated by means of electron microscopy. The labelling of fibrillary actin underlines the cellular heterogeneity and the different cellular functional state of the tumor cells. These findings are interpreted as an evidence of a multidirectional differentiation potential of the gonadal stroma mesenchyme. These observations support the classification of these neoplasia as gonadal stromal tumors particularly in light of the clearly visible differentiation. Problems of evaluating the malignant potential of gonadal stromal tumors are also discussed.

Actin Cytoskeleton↗
PubMed1992

Signal enhancement in fluorescence microscopy and flow cytometry using fluorescent liposome-antibody conjugates as second layer reagents.

We have developed small unilamellar liposomes (SUV) containing carboxyfluorescein for indirect immunophenotyping of cells by fluorescent microscopy and flow cytometry. Covalently coupled to anti-mouse Ig and anti-human Ig with mouse monoclonal antibodies and human typing antisera respectively, these conjugates can increase sensitivity by a factor of 10 compared to fluorescein isothiocyanate (FITC) conjugates. No increase in background fluorescence was observed. The reagents have been used to detect a low density antigen, the interleukin-2 receptor, on resting T-cells and to overcome the quenching effect of haemoglobin in fluorescent phenotyping of red cells. This has allowed to accurately estimate blood grouping in mixed fields and with small numbers of cells. We have determined the blood group of fetal red cells obtained from a chorionic villous sample at 10 weeks gestation and predicted severe hemolytic disease. This was confirmed by fetal sampling at 19 weeks and several intra-uterine transfusions were undertaken before a successful delivery at 36 weeks. Applications of the method in leucocyte phenotyping and transfusion medicine are discussed.

Animals↗
PubMed1997

Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy.

Imaging of single fluorescent molecules has been achieved in a relatively simple manner using objective-type total internal reflection fluorescence microscopy (TIRFM). Switching from epi-fluorescence microscopy to objective-type TIRFM was achieved by translation of a single mirror in the system. Clear images of single molecules of an orange fluorescent dye, Cy3, were obtained with a fluorescence-to-background ratio of 12, using a conventional high aperture objective (PlanApo, 100 x, NA 1.4) with ordinary coverslips and immersion oil. This method allowed visualization of single molecules under scanning probe microscopes. Taking advantage of the technique of single molecule imaging, individual ATP turnovers have been visualized with a fluorescent ATP analogue, Cy3-ATP, using a simple experimental strategy. Clear on/off signals were obtained that correspond to the association and dissociation of single Cy3-ATP/ADP molecules with a single myosin head molecule. This method will allow a variety of single-molecular assays of biomolecular functions to be performed using fluorescently labeled substrates, ligands, messengers, and biologically active molecules. Thus, the present technique provides a simple yet powerful and universal tool for researchers to probe the events of single molecules.

Adenosine Triphosphatases↗
PubMed2000

[Stereologic analysis of vacuolization of the T-system of frog muscle fibers, detected using confocal fluorescence microscopy].

The confocal fluorescence microscopy has been used for quantitative evaluation of the T-system reversible vacuoles produced by efflux of 80-120 mM glycerol from frog skeletal muscle fibers. The fibers were stained by membrane probe RH414 and by water-soluble dye fluorescein dextran that marks the vacuolar lumen. Using morphometrical and stereological methods the volume and surface densities of vacuoles were measured on single optical sections and Z-series during a 30 min glycerol efflux. Various methods of measurements (three-dimensional reconstruction of vacuoles, computer morphometry, point counting method) applied to the same Z-series provide similar results. The vacuolar membranes stained by RH414 look like bright rings 0.3-0.4 micron in width. It is concluded that the real position of vacuolar membrane corresponds to the middle of the vacuolar envelope. The measurements of the external dimensions of the envelope overestimate the stereological parameters up to 50%. The volume density of vacuoles reaches 10% within 20-30 min of glycerol efflux. It means that the volume of the T-system may increase by 25-30 times compared to the control value (0.3-0.4%). The surface density of vacuoles during reversible vacuolation is equal to 0.20-0.35 micron-1 and does not exceed the surface density of normal T-system. The sufficiency of membrane material for the T-system reversible vacuolation is discussed in addition to the role of geometrical factor in this phenomenon.

Animals↗
PubMed1981

Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy.

Permeant cationic fluorescent probes are shown to be selectively accumulated by the mitochondria of living cells. Mitochondria-specific interaction of such molecules is apparently dependent on the high trans-membrane potential (inside negative) maintained by functional mitochondria. Dissipation of the mitochondrial trans-membrane and potential by ionophores or inhibitors of electron transport eliminates the selective mitochondrial association of these compounds. The application of such potential-dependent probes in conjunction with fluorescence microscopy allows the monitoring of mitochondrial membrane potential in individual living cells. Marked elevations in mitochondria-associated probe fluorescence have been observed in cells engaged in active movement. This approach to the analysis of mitochondrial membrane potential should be of value in future investigations of the control of energy metabolism and energy requirements of specific biological functions at the cellular level.

Animals↗
PubMed1993

Dynamic measurements of intracellular aminopeptidase activity in hepatocytes using multiparameter digitized video fluorescent microscopy.

Digitized video fluorescent microscopy (DVFM) is a powerful technique for quantitating multiple processes in living cells. However, techniques for measuring protease activity by DVFM are not available. Our aim was to develop an approach for measuring aminopeptidase activity using DVFM. We conjugated glycine-7-amino-4-methylcoumarin-3-acetic acid (glycine-AMC-3-acetic acid) to dextran using a PEG bridge. Glycine-AMC-3-acetic acid-PEG-dextran was microinjected into cultured rat hepatocytes along with rhodamine-dextran. Glycine-AMC-3-acetic acid-PEG-dextran is nonfluorescent, but aminopeptidase hydrolysis of the glycine-AMC bond liberates the fluorescent AMC-3-acetic acid-PEG-dextran within the cell. Following microinjection, rhodamine-dextran fluorescence remained constant while AMC-3-acetic acid-PEG-dextran fluorescence increased in a linear fashion over time reflecting proteolytic cleavage of the glycine-AMC bond. AMC-3-acetic acid-PEG-dextran and rhodamine-dextran fluorescence were cytosolic as evidenced by diffuse fluorescence and colocalized. Because rhodamine-dextran fluorescence remained constant and the probes colocalized, the fluorescent ratio of AMC-3-acetic acid-PEG-dextran/rhodamine-dextran could be used to measure proteolysis. Basal rates of proteolysis were 9 +/- 3 ratio units/10 min. Comicroinjection of the aminopeptidase inhibitor, bestatin, along with the dextran probes abolished proteolysis. Addition of the calcium ionophore, 4-Br-A23187, increased proteolysis 12-fold to 107 +/- 14/10 min (P < 0.01). We have developed a novel, dynamic technique for measuring pH-sensitive, Ca(2+)-dependent aminopeptidase activity in living cells using DVFM. This approach may be used for the measurement of other peptidase activities by synthesizing peptidase-specific peptidyl-AMC-3-acetic acid-PEG-dextran conjugates.

Aminopeptidases↗
PubMed2005

Two-point separation in far-field super-resolution fluorescence microscopy based on two-color fluorescence dip spectroscopy, Part I: Experimental evaluation.

The two-point resolution of a novel two-color far-field super-resolution fluorescence microscopy was evaluated by measuring fluorescent beads 100 nm in diameter. This microscopy is based on a combination of two-color fluorescence dip spectroscopy and a phase-modulation technique for a laser beam. By simply introducing two-color laser light, the size of the fluorescent image of a bead was shrunk down to a diameter of 250 nm from the diffraction-limited image with a diameter of 360 nm. For two closely adjacent fluorescent beads with a separation distance of 350 nm, the two-color microscope clearly gave separated fluorescence images, while the conventional one-color fluorescence microscope could not resolve them. It has been proved that our technique breaks Rayleigh's diffraction limit.

Fluorescent Dyes↗
PubMed1998

Electron and fluorescence microscopy of extracellular glucan and aryl-alcohol oxidase during wheat-straw degradation by Pleurotus eryngii.

The ligninolytic fungus Pleurotus eryngii grown in liquid medium secreted extracellular polysaccharide (87% glucose) and the H2O2-producing enzyme aryl-alcohol oxidase (AAO). The production of both was stimulated by wheat-straw. Polyclonal antibodies against purified AAO were obtained, and a complex of glucanase and colloidal gold was prepared. With these tools, the localization of AAO and extracellular glucan in mycelium from liquid medium and straw degraded under solid-state fermentation conditions was investigated by transmission electron microscopy (TEM) and fluorescence microscopy. These studies revealed that P. eryngii produces a hyphal sheath consisting of a thin glucan layer. This sheath appeared to be involved in both mycelial adhesion to the straw cell wall during degradation and AAO immobilization on hyphal surfaces, with the latter evidenced by double labelling. AAO distribution during differential degradation of straw tissues was observed by immunofluorescence microscopy. Finally, TEM immunogold studies confirmed that AAO penetrates the plant cell wall during P. eryngii degradation of wheat straw.

Alcohol Oxidoreductases↗
PubMed1985

Immunogold probes for electron microscopy: evaluation of staining by fluorescence microscopy.

A method is presented whereby the staining of intracellular structures with immunogold probes for electron microscopy can be evaluated at the light microscopic level. Methanol-fixed monolayers of cultured Dunning R-3327-H rat prostatic adenocarcinoma cells were stained for cytokeratins using a two-step immunogold technique consisting of primary anti-keratin antibody followed by gold-labeled secondary antibody. Bound immunogold probe was then visualized with a fluorescent tertiary anti-immunogold probe antibody. Fluorescence microscopy of the whole cell monolayers showed a typical keratin cytoskeleton. The extra staining step did not interfere with subsequent fixation, embedding, and sectioning for electron microscopy, which showed cytoplasmic intermediate filaments decorated with colloidal gold. Using this method, it should be possible to manipulate parameters critical to staining with immunogold probes and to evaluate the labeling without necessitating repeated time-consuming electron microscopic processing. The method also provides a useful correlation between the light microscopic and ultrastructural labeling patterns of immunogold probes.

Adenocarcinoma↗
PubMed2004

Insertion of GPI-anchored alkaline phosphatase into supported membranes: a combined AFM and fluorescence microscopy study.

A new method based on combined atomic force microscopy (AFM) and fluorescence microscopy observations, is proposed to visualize the insertion of glycosylphosphatidyl inositol (GPI) anchored alkaline phosphatase from buffer solutions into supported phospholipid bilayers. The technique involves the use of 27 nm diameter fluorescent latex beads covalently coupled to the amine groups of proteins. Fluorescence microscopy allows the estimation of the relative protein coverage into the membrane and also introduces a height amplification for the detection of protein/bead complexes with the AFM. The coupling of the beads with the amine groups is not specific; this new and simple approach opens up new ways to investigate proteins into supported membrane systems.

Alkaline Phosphatase↗
PubMed2006

High-resolution solid modeling of biological samples imaged with 3D fluorescence microscopy.

Optical-sectioning, digital fluorescence microscopy provides images representing temporally- and spatially-resolved molecular-scale details of the substructures of living cells. To render such images into solid models for further computational analyses, we have developed an integrated system of image acquisition, processing, and rendering, which includes a new empirical technique to correct for axial distortions inherent in fluorescence microscopy due to refractive index mismatches between microscope objective immersion medium, coverslip glass, and water. This system takes advantage of the capabilities of ultra-high numerical aperture objectives (e.g. total internal reflection fluorescence microscopy) and enables faithful three-dimensional rendering of living cells into solid models amenable to further computational analysis. An example of solid modeling of bovine aortic endothelial cells and their nuclei is presented. Since many cellular level events are temporally and spatially confined, such integrated image acquisition, processing, rendering, and computational analysis, will enable, in silico, the generation of new computational models for cell mechanics and signaling.

Animals↗
PubMed2006

In vivo imaging of the bronchial wall microstructure using fibered confocal fluorescence microscopy.

RATIONALE: Fibered confocal fluorescence microscopy (FCFM) is a new technique that produces microscopic imaging of a living tissue through a 1-mm fiberoptic probe that can be introduced into the working channel of the bronchoscope. OBJECTIVES: To analyze the microscopic autofluorescence structure of normal and pathologic bronchial mucosae using FCFM during bronchoscopy. METHODS: Bronchial FCFM and spectral analyses were performed at 488-nm excitation wavelength on two bronchial specimens ex vivo and in 29 individuals at high risk for lung cancer in vivo. Biopsies of in vivo FCFM-imaged areas were performed using autofluorescence bronchoscopy. RESULTS: Ex vivo and in vivo microscopic and spectral analyses showed that the FCFM signal mainly originates from the elastin component of the basement membrane zone. Five distinct reproducible microscopic patterns were recognized in the normal areas from the trachea down to the more distal respiratory bronchi. In areas of the proximal airways not previously biopsied, one of these patterns was found in 30 of 30 normal epithelia, whereas alterations of the autofluorescence microstructure were observed in 19 of 22 metaplastic or dysplastic samples, five of five carcinomas in situ, and two of two invasive lesions. Disorganization of the fibered network could be found on 9 of 27 preinvasive lesions, compatible with early disruptions of the basement membrane zone. FCFM alterations were also observed in a tracheobronchomegaly syndrome and in a sarcoidosis case. CONCLUSIONS: Endoscopic FCFM represents a minimally invasive method to study specific basement membrane alterations associated with premalignant bronchial lesions in vivo. The technique may also be useful to study the bronchial wall remodeling in nonmalignant chronic bronchial diseases.

Bronchi↗
PubMed2006

Latex diffusion at high volume fractions studied by fluorescence microscopy.

The behavior of fluorescent latex probes (radii 0.05, 0.1, and 0.5 mum) in latex host particle suspensions was investigated by fluorescence microscopy with image analysis. The volume fraction of the host latex was varied between 0 and 0.50. A careful statistical analysis was performed to examine the accuracy of the fluorescence microscopy method, from which the direct observation of the Brownian motion gives the diffusion coefficient. The method was found to meet all statistical requirements. From rheological measurements, the maximum volume fraction and the intrinsic viscosity can be obtained. The Krieger-Dougherty equation can be used for the prediction of sample viscosities. The predicted viscosities were used to obtain the theoretical diffusion coefficients with the Stoke-Einstein equation. When comparing the theoretical diffusion coefficients with the experimental ones, it turned out that all models tested yielded acceptable predictions of the diffusion coefficients.

Journal Article↗
PubMed2006

The sampling limit in fluorescence microscopy.

Sampling in fluorescence microscopy is treated using the concept of the three-dimensional (3D) optical transfer function (OTF). The border of the OTF frequency surface defines the required minimum sampling. The shape of the OTF is derived from simple considerations and valid for far-field high numerical aperture, vector theory. Optimal regular sampling is achieved by a hexagonal grid in 2D, and corresponding hexagonal structures, body-centered cubic (bcc) and hexagonal close-packed (hcp) structures, in 3D. As compared to standard (rectilinear grid) sampling a reduction of 13.4% in 2D and 29.3% in 3D can be achieved with optimized sampling. This reduction in data size is also accompanied by an imaging speed improvement, a reduction of sample bleaching, and can lead to imaging with better signal to noise ratio.

Journal Article↗
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↗