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Results for “Fluorescence Microscopy”

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At least 1,225 records · Page 68Linked to original sources
PubMed1985

The use of FITC-conjugated monoclonal antibodies for determination of S-phase cells with fluorescence microscopy.

A method is modified to determine the DNA synthesizing cells in primary human breast tumors and cells with idiopathic thrombocytopenic purpura (ITP) with FITC-conjugated monoclonal antibody against bromodeoxyuridine (FITC-M-anti-BrdUrd) and fluorescence microscopy. The DNA synthesizing cells were also determined from a portion of the same tissues by classical tritiated thymidine labeling (3HdThd) and autoradiography. The results from bromodeoxyuridine labeling index (BrdUrd-LI) and tritiated thymidine labeling index (3HdThd-LI) obtained from the same tissues were compared. The mean BrdUrd-LI for breast tumor was 5.4 +/- 1.0% and the mean 3HdThd-LI was 5.5 +/- 1.1%. Similarly, the labeling indexes obtained from mononuclear leukocytes of healthy donors had means of 0.5 +/- 0.1% and 0.6 +/- 0.1% for BrdUrd-LI and 3HdThd-LI, respectively. The change in the proliferation rate of mononuclear leukocyte population in the samples obtained from patients with ITP could be observed by both methods. The mean BrdUrd-LI of mononuclear leukocytes for this hematological disorder was 5.4 +/- 0.8%. These results suggest that was 6.1 +/- 0.8%. These results suggest that this relatively simple technique offers an alternative method for determining the DNA synthesizing cells in a given cell population.

Antibodies, Monoclonal↗
PubMed2001

Breaking Abbe's diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes.

We report on the generation of various hole-centered beams in the focal region of a lens and investigate their effectiveness to break the diffraction barrier in fluorescence microscopy by stimulated emission. Patterning of the phase of the stimulating beam across the entrance pupil of the objective lens produces point-spread-functions with twofold, fourfold, and circular symmetry, which narrow down the focal spot to 65-100 nm. Comparison with high-resolution confocal images exhibits a resolution much beyond the diffraction barrier. Particles that are only 65-nm apart are resolved with focused light.

Journal Article↗
PubMed2002

Resolution improvement in two-photon fluorescence microscopy with a single-mode fiber.

The dependence of spectral broadening of an ultrashort-pulsed laser beam on the fiber length and the illumination power is experimentally characterized in order to deliver the laser for two-photon fluorescence microscopy. It is found that not only the spectral width but also the spectral blue shift increases with the fiber length and illumination power, owing to the nonlinear response in the fiber. For an illumination power of 400 mW in a 3-m-long single-mode fiber, the spectral blue shift is as large as 15 nm. Such a spectral blue shift enhances the contribution from the short-wavelength components within the pulsed beam and leads to an improvement in resolution under two-photon excitation, whereas the efficiency of two-photon excitation is slightly reduced because of the temporal broadening of the pulsed beam. The experimental measurement of the axial response to a two-photon fluorescence polymer block confirms this feature.

Journal Article↗
PubMed1995

Simultaneous measurement of [Ca2+]i and secretion-coupled membrane turnover, by single cell fluorescence microscopy.

Thyrotropin releasing hormone (TRH), which stimulates prolactin secretion, increases the fluorescence of cultured bovine anterior pituitary (bAP) cells in the presence of the non-permeant membrane indicator dye FM 1-43 [Stafford SJV. Shorte SL. Schofield JG. (1993) Use of a fluorescent dye to measure secretion from intact bovine anterior pituitary cells. Biosci. Rep., 13, 9-17]. FM 1-43 is non-fluorescent in aqueous solution but becomes fluorescent when incorporated into the plasma membrane. The membrane area accessible to FM 1-43 dye, and therefore cell fluorescence, increases during exocytosis as secretory granules fuse with the plasma membrane, and endocytosis as vesicles formed at the plasma-membrane fuse with intracellular organelle membranes. We have here measured changes in FM 1-43 uptake and the intracellular calcium concentration ([Ca2+]i) concurrently in the same cells on exposure to TRH, phorbol myristate acetate (PMA) or NH4Cl. TRH (0.1-10 microM) caused a transient increase in [Ca2+]i in 70-90% of bAP cells and in 60-90% of the responding cells also caused a sustained increased FM 1-43 fluorescence. TRH increased [Ca2+]i but did not affect FM 1-43 fluorescence in GH3 rat pituitary cells, probably because they contain too few secretory granules to give a detectable increase. The dopamine D2-receptor agonist quinpirole (10 microM) had little effect on the TRH-induced [Ca2+]i rise in bAP cells, but abolished the increase in FM 1-43 fluorescence. The phorbol ester PMA (0.3-3 microM) caused a small, transient increase in [Ca2+]i followed by a fall to levels lower than original resting levels in 40-60% of bAP cells and increased FM 1-43 uptake in cells showing these changes. Extracellular NH4Cl, which mobilises calcium from an ionomycin-insensitive calcium store, caused a transient [Ca2+]i increase in over 90% of the bAP-cells and increased FM 1-43 uptake in a subpopulation (> 50%) of these. The Na+/H+ ionophore monensin prevented the increase in FM 1-43 fluorescence but not the [Ca2+]i rise induced by TRH, prevented the increases in both FM 1-43 fluorescence and [Ca2+]i caused by NH4Cl, and reduced the number of cells showing a rise in FM 1-43 fluorescence in response to PMA from 64% to 34%. The Ca(2+)-ATPase inhibitor thapsigargin reduced the number of bAP cells displaying TRH-induced increases in [Ca2+]i and membrane-turnover from 74% to 18%, but did not affect the changes in [Ca2+]i or FM 1-43 fluorescence caused by PMA or NH4Cl. We discuss the relationships between the secretogogue-induced increases in FM 1-43 fluorescence and changes in intracellular [Ca2+]i under these conditions.

Animals↗
PubMed1982

[Structural changes in muscle fiber contractile proteins studied by polarization ultraviolet fluorescence microscopy. VI. Conformational restructurings of F-actin induced by the binding of heavy meromyosin].

Decrease in tryptophan fluorescence anisotropy of a single ghost muscle fibre was found at the binding of heavy meromyosin (HMM) to actin. Polarized fluorescence revealed its peak changes at a molar ratio of HMM/actin equal to 0.1. The changes observed in polarized fluorescence at F-actin-HMM interaction were found to depend on the state of HMM. The changes in anisotropy fluorescence under the same conditions were assumed to be independent of tryptophane residues in HMM, reflecting cooperative changes in F-actin conformation. Changes in the conformation of F-actin are of great importance in muscular contraction.

Actins↗
PubMed2001

Spontaneous and MNNG-induced reversion of an EGFP construct in HeLa cells: an assay for observing mutations in living cells by fluorescent microscopy.

A HeLa cell line stably expressing the enhanced green fluorescence protein (EGFP) gene, interrupted by the HBB IVS2-654 intron, was studied without treatment and after treatment with a single standard dose of 15 microM of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This assay was done in order to prove that such a construct can revert by a variety of mechanisms and that it produces a visible phenotype, i.e., green fluorescence. The system permits visual detection of living mutant cells among a background of non-mutant cells and does not require a selective medium. The results show that the construct reverts by large deletions (-62, -100, and -162 bp), small insertions (+4 bp), small rearrangements (19 bp duplication), base substitutions at purines (G652, G653, A655, G579), and a pyrimidine (T654) between nucleotide positions 579 and 837. Splice-site mutations were recovered, and some of the mechanisms underlying these mutations are discussed. Because of the ease of detection of revertant cells under fluorescent light and the wide variety of mutations that can be recovered, further development of this system could make it a useful new mammalian cell mutagenicity assay.

Base Sequence↗
PubMed1983

Demonstration of early lesions of muscle by infrared fluorescence microscopy.

Previous investigations demonstrated decrease or loss of IR fluorescence in certain lesions of smooth and striated muscle. These investigations were extended to diseases of skeletal muscle. Sections were stained and photographed as described (Puchtler et al. 1980). Various diseases caused significant changes of IR fluorescence. Often, IR fluorescence demonstrated lesions in muscle fibers which appeared unremarkable in direct light and/or conventional darkfield microscopy. The patterns of IR fluorescence varied widely, even within groups of diseases, e.g. muscular dystrophies. However, owing to the heterogeneity of the available material, it is not yet possible to draw conclusions concerning the diagnostic significance of different IR fluorescence patterns. Hence this report should be regarded only as a pilot study.

Adolescent↗