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 1,279 records · Page 71Linked to original sources
PubMed2002

Focal spots of size lambda/23 open up far-field fluorescence microscopy at 33 nm axial resolution.

We report spots of excited molecules of 33 nm width created with focused light of lambda = 760 nm wavelength and conventional optics along the optic axis. This is accomplished by exciting the molecules with a femtosecond pulse and subsequent depletion of their excited state with red-shifted, picosecond-pulsed, counterpropagating, coherent light fields. The lambda/23 ratio constitutes what is to our knowledge the sharpest spatial definition attained with freely propagating electromagnetic radiation. The sub-diffraction spots enable for the first time far-field fluorescence microscopy with resolution at the tens of nanometer scale, as demonstrated in images of membranes of bacillus megaterium.

Journal Article↗
PubMed1991

The three-dimensional architecture of the mitotic spindle, analyzed by confocal fluorescence and electron microscopy.

Fluorescence microscopy techniques have become important tools in mitosis research. The well-known disadvantages of fluorescence microscopy, rapid bleaching, phototoxicity and out-of-focus contributions blurring the in-focus image are obstacles which still need to be overcome. Confocal fluorescence microscopy has the potential to improve our capabilities of analyzing cells, because of its excellent depth-discrimination and image processing power. We have been using a confocal fluorescence microscope for the study of the mechanism of poleward chromosome movement, and report here 1) a cell preparation technique, which allows labeling of fixation sensitive spindle antigens with acceptable microtubule preservation; 2) the use of image processing methods to represent the spatial distribution of various labeled elements in pseudocolour; 3) a novel immunoelectron microscopic labeling method for microtubules, which allows the visualization of their distribution in semithin sections at low magnification; and 4) a first attempt to study microtubule dynamics with a confocal fluorescence microscope in living cells, microinjected with rhodamine labeled tubulin. Our experience indicates that confocal fluorescence microscopy provides real advantages for the study of spatial colocalization of antigens in the mitotic spindle. It does not, however, overcome the basic limits of resolution of the light microscope. Therefore, it has been necessary to use an electron microscopic method. Our preliminary results with living cells show that it is possible to visualize the entire microtubule network in stereo, but that the sensitivity of the instrument is still too low to perform dynamic time studies. It will be worthwhile to further develop this new type of optical instrumentation and explore its usefulness on both fixed and living cells.

Animals↗
PubMed1998

Continuous wave excitation two-photon fluorescence microscopy exemplified with the 647-nm ArKr laser line.

We report on efficient two-photon fluorescence imaging in beam scanning microscopy by exciting UV dyes at the 647-nm line of a continuous wave ArKr mixed gas laser. For a numerical aperture of 1.4 (oil), we used an illumination power of up to 210 mW at the sample. High-resolution images were obtained for DAPI-labelled cell nuclei within 4-60 s. Our method is a simple two-photon alternative to UV confocal imaging with the potential of becoming a very useful feature of laser scanning microscopy.

Animals↗
PubMed2003

Cellular events preceding acetaminophen cataractogenesis studied by confocal fluorescence microscopy.

Acetaminophen (APAP) is biotransformed by hepatic cytochrome P450 (CYP) enzymes to the cataractogenic metabolite N-acetyl-p-benzoquinone imine (NAPQI). In the previous studies in which NAPQI was injected into the anterior chamber of mouse eye, we observed mitochondrial dysfunction and disturbances in Ca2+ homeostasis in the lens epithelium, and activation of the nonlysosomal neutral protease calpain. In this work we investigated whether intraperitoneal injection of APAP elicits similar cellular responses in the lens epithelium prior to the onset of lens opacity development. Following APAP injection, reactive oxygen species generation, intracellular free Ca2+ increase and calpain activation in the lens epithelium were determined in situ by fluorescence confocal microscopy. It was found that cellular events in the lens prior to the onset of opacification were essentially identical to those elicited by NAPQI. In addition, lens calpain activities were characterized based on their Ca2+ requirement and several calpain inhibitors were shown to prevent cataract development.

Acetaminophen↗
PubMed1995

Implementation of an NSOM system for fluorescence microscopy.

We describe our progress toward an NSOM system intended for fluorescence imaging of biological samples. This process included integration of shear-force feedback into an existing NSOM system. Topographic images acquired using uncoated tips are presented. We also present our initial effort at simultaneous acquisition of topographic and fluorescence data using an aluminum coated tip.

Equipment Design↗
PubMed1998

[Fluorescence microscopy for detection of frauds in ground coffee].

Currently, frauds in ground coffee are detected with traditional microscopy, which is a tedious and not very precise method. Therefore, a more efficient and rapid microscopical method based on fluorescence, was proposed. For the microscopy analysis, pure coffee was compared to coffee samples adulterated with rye, barley, corn and wheat at 2.5, 5.0, and 10.0% levels. Starch granules were detected and identified immediately with fluorescence microscopy, in the adulterated coffee mixtures, quite different from optical microscopy where no frauds were detected with certainty. The suggested fluorescence method for the detection of cereal frauds in coffee was found to be more efficient and precise than the current official one.

Coffee↗
PubMed1985

Digital imaging fluorescence microscopy: spatial heterogeneity of photobleaching rate constants in individual cells.

Photobleaching and related photochemical processes are recognized experimental barriers to quantification of fluorescence by microscopy. We have measured the kinetics of photobleaching of fluorophores in living and fixed cells and in microemulsions, and have demonstrated the spatial variability of these processes within individual cells. An inverted fluorescence microscope and a high-sensitivity camera, together with high-speed data acquisition by a computer-controlled image processor, have been used to control precisely exposure time to excitation light and to record images. To improve the signal-to-noise ratio, 32 digital images were integrated. After correction for spatial variations in camera sensitivity and background fluorescence, the images of the relative fluorescence intensities for 0.065 micron2 areas in the object plane were obtained. To evaluate photobleaching objectively, an algorithm was developed to fit a three-parameter exponential equation to 20 images recorded from the same microscope field as a function of illumination time. The results of this analysis demonstrated that the photobleaching process followed first-order reaction kinetics with rate constants that were spatially heterogeneous and varied, within the same cell, between 2- and 65-fold, depending on the fluorophore. The photobleaching rate constants increased proportionally with increasing excitation intensity and, for benzo(a)pyrene, were independent of probe concentration over three orders of magnitude (1.25 microM to 1.25 mM). The propensity to photobleach was different with each fluorophore. Under the cellular conditions used in these studies, the average rates of photobleaching decreased in this order: N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol greater than acridine orange greater than rhodamine-123 greater than benzo(a)pyrene greater than fluorescein greater than tetramethylrhodamine greater than 1,1'dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine. The photobleaching appears to be an oxidation reaction, in that the addition of saturated solutions of Na2S2O5 to mineral oil microemulsions eliminated photobleaching of N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol or benzo(a)pyrene. We identified experimental conditions to observe, without detectable photobleaching, fluorophores in living cells, which can not be studied anaerobically. Useful images were obtained when excitation light was reduced to eliminate photobleaching, as determined from zero-time images calculated from the exponential fit routine.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Chloro-7-nitrobenzofurazan↗
PubMed2000

An efficient algorithm for measurement and correction of chromatic aberrations in fluorescence microscopy.

Even the best optical microscopes available on the market exhibit chromatic aberrations to some extent. In some types of study, chromatic aberrations of current optics cannot be neglected and a software correction is highly desirable. This paper describes a novel method of chromatic aberration measurement and software correction using sub-resolution bead imaging and computer image analysis. The method is quick, precise and enables the determination of both longitudinal and lateral chromatic aberrations. Correction function can be computed in about half an hour, including image acquisition. Using this approach, chromatic aberrations can be reduced to 10-20 nm laterally and 10-60 nm axially depending on the type of optical set-up. The method is especially suitable for fluorescence microscopy, where a limited number of wavelengths are observed.

Journal Article↗
PubMed1999

Telomere length measurements using digital fluorescence microscopy.

BACKGROUND: The ends of chromosomes (telomeres) are important to maintain chromosome stability, and the loss of telomere repeat sequences has been implicated in cellular senescence and genomic instability of cancer cells. The traditional method for measuring the length of telomeres (Southern analysis) requires a large number of cells (>10(5)) and does not provide information on the telomere length of individual chromosomes. Here, we describe a digital image microscopy system for measurements of the fluorescence intensity derived from telomere repeat sequences in metaphase cells following quantitative fluorescence in situ hybridization (Q-FISH). METHODS: Samples are prepared for microscopy using Q-FISH with Cy3 labeled peptide nucleic acid probes specific for (T(2)AG(3))(n) sequences and the DNA dye DAPI. Separate images of Cy3 and DAPI fluorescence are acquired and processed with a dedicated computer program (TFL-TELO). With the program, the integrated fluorescence intensity value for each telomere, which is proportional to the number of hybridized probes, is calculated and presented to the user. RESULTS: Indirect tests of our method were performed using simulated as well as defined tests objects. The precision and consistency of human telomere length measurements was then analyzed in a number of experiments. It was found that by averaging the results of less than 30 cells, a good indication of the telomere length (SD of 10-15%) can be obtained. CONCLUSIONS: We demonstrate that accurate and repeatable fluorescence intensity measurements can be made from Q-FISH images that provide information on the length of telomere repeats at individual chromosomes from limited number of cells.

Algorithms↗
PubMed2000

Single long DNA molecule analysis using fluorescence microscopy.

Single long DNA molecule (T4 DNA) in agarose gel was visualized with a fluorescence microscope. We confirmed alternating current electric fields is effective for stretching of single DNA molecule in agarose gel. This stretching phenomenon was observed with wide range of agarose gel concentration from 0.5%(W/V) to 1.5%. From this observation, the presence of agarose gel fiber is essential for this stretching phenomenon. The stretching process of several DNA molecules in gel shows discontinuity, which is never observed in polymer systems. It would be based on topological restriction from gel fibers.

Bacteriophage T4↗
PubMed1995

The cytologic diagnosis of Mycobacterium kansasi tuberculosis by fluorescence microscopy of Papanicolaou-stained specimens.

The sensitivities of (i) Papanicolaou fluorescence, (ii) auramine rhodamine fluorescence, and (iii) Ziehl-Neelsen staining were compared for their ability to detect the atypical mycobacterium Myco. kansasi in cytological samples. Ninety-two cases were investigated, and the sensitivities of the three methods of detection were found to be 36.9%, 12.0%, and 20.7% respectively. The control groups consisted of 30 specimens from cases of bronchial carcinoma and 30 of pneumonia. All cases were proved by microbiology. No false-positive results were recorded using Papanicolaou fluorescence. An important but coincidental finding arising from this study was that infection by the atypical mycobacterium Myco. kansasi causes cytological patterns corresponding to those normally associated with acute pneumonia and not to tuberculosis.

Adult↗
PubMed2002

A new high-aperture glycerol immersion objective lens and its application to 3D-fluorescence microscopy.

High-resolution light microscopy of glycerol-mounted biological specimens is performed almost exclusively with oil immersion lenses. The reason is that the index of refraction of the oil and the cover slip of approximately 1.51 is close to that of approximately 1.45 of the glycerol mountant, so that refractive index mismatch-induced spherical aberrations are tolerable to some extent. Here we report the application of novel cover glass-corrected glycerol immersion lenses of high numerical aperture (NA) and the avoidance of these aberrations. The new lenses feature a semi-aperture angle of 68.5 degrees, which is slightly larger than that of the diffraction-limited 1.4 NA oil immersion lenses. The glycerol lenses are corrected for a quartz cover glass of 220 microm thickness and for a 80% glycerol-water immersion solution. Featuring an aberration correction collar, the lens can adapt to glycerol concentrations ranging between 72% and 88%, to slight variations of the temperature, and to the cover glass thickness. As the refractive index mismatch-induced aberrations are particularly important to quantitative confocal fluorescence microscopy, we investigated the axial sectioning ability and the axial chromatic aberrations in such a microscope as well as the image brightness as a function of the penetration depth. Whereas there is a significant decrease in image brightness associated with oil immersion, this decrease is absent with the glycerol immersion system. In addition, we show directly the compression of the optic axis in the case of oil immersion and its absence in the glycerol system. The unique advantages of these new lenses in high-resolution microscopy with two coherently used opposing lenses, such as 4 Pi-microscopy, are discussed.

Animals↗
PubMed2005

The use of fluorescence microscopy to visualise homotypic interactions of tomato spotted wilt virus nucleocapsid protein in living cells.

Fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) were employed to study homotypic protein-protein interactions in living cells. To this end, the nucleocapsid (N) protein of tomato spotted wilt virus (TSWV) was expressed as a fusion protein with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP). Co-expression experiments of the two fusion proteins were carried out in baby hamster kidney (BHK21) cells. Both the wild type and the fusion proteins showed a peri-nuclear localisation pattern and were observed to form aggregates. In sensitised emission experiments, energy transfer was observed to take place from CFP to YFP when the two fluorophores were fused to TSWV N protein, indicating strongly homotypic interaction of the N proteins. This was confirmed by acceptor photobleaching studies as well as by FLIM experiments. All three methods showed interactions taking place, not only in the aggregates in the peri-nuclear region, but also throughout the cytoplasm. These experiments clearly demonstrated the potential of these fluorescence methods for studying the interactions of viral proteins in living cells.

Animals↗