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At least 1,387 records · Page 77Linked to original sources
PubMed2002

Direct visualization of abasic sites on a single DNA molecule using fluorescence microscopy.

A new method was developed to allow direct visualization of damaged sites on individual DNA molecules. Fluorescence in situ hybridization on extended DNA molecules was modified to detect a single abasic site. Abasic sites were specifically labeled with a biotinylated aldehyde-reactive probe and fluorochrome-conjugated streptavidin. The light emitted by a single fluorochrome-DNA complex was calibrated. The number of abasic sites on the DNA molecule was estimated by counting each fluorochrome-DNA complex. The present study directly visualized and characterized the abasic sites of single DNA molecules.

DNA↗
PubMed1990

Fluorescence microscopy of the dynamics of supercoiling, folding, and condensation of bacterial chromosomes, induced by acridine orange.

The fluorescent dye, acridine orange, was used to visualize bacterial chromosomes extending from bacteria attached to a glass surface. The acridine-induced condensation of these chromosomes was followed in real-time with a low light level video camera. Acridine orange induced the packing of the bacterial chromosome into thick bundles which underwent various forms of condensation, supercoiling, folding, and rolling into a compact particle. Filaments attached to the surface at both ends were topologically constrained and supercoiled rapidly; whereas all three patterns of condensation were noted among filaments attached at only one end or free from the surface. Kinks often appeared in the filaments prior to supercoiling or folding, and the dynamic events observed often occurred around these kinks. These observations identify several mechanisms of condensation available to higher order structures of DNA, and indicate that kinks are an important intermediate step in many of the transitions.

Acridine Orange↗
PubMed2000

Myosin monomer density and exchange in synthetic thick filaments investigated using fluorescence microscopy with single molecule sensitivity.

The number of myosin molecules in synthetic thick filaments, prepared by dialysis at 0.12 M NaCl and pH 7.0, was estimated to be between 400 and 800 molecules per micrometre under conditions appropriate for in vitro motility assays. This estimate was based on a number count of Cy3-labelled myosin molecules incorporated into filaments at a nominal ratio of 1:1000. At this dilution, single fluorescent spots were resolved corresponding to individual labelled myosins. The spots usually bleached with a one- or two-step profile but, in around 30% of the cases, fluctuations were observed indicating that additional photophysical or photochemical events had occurred. Myosin molecules were shown to exchange between filaments in suspension on a time-scale of several hours at 4 degrees C, but the reaction was only 75% complete after 48 h, suggesting a non-exchangeable core. However, myosin exchange does not appear to be the predominant source of the fluctuations in fluorescence intensity in the single molecule assays.

Adenosine Triphosphate↗
PubMed1988

Enumeration of IFN-gamma-producing cells by flow cytometry. Comparison with fluorescence microscopy.

A new intracytoplasmic immunofluorescence staining to detect and quantify human interferon-gamma (IFN-gamma)-producing cells by means of flow cytometry is described. Mononuclear leukocytes, stimulated in vitro to produce IFN-gamma, were fixed and made permeable to antibodies by sequential exposure to paraformaldehyde and the detergent n-octyl-glucoside. Cytoplasmic IFN-gamma was demonstrated by indirect immunofluorescence using IFN-gamma-specific mouse monoclonal antibodies. The staining exhibited a very characteristic morphology and was localized in the Golgi apparatus. An excellent agreement between the enumeration of cytoplasmic IFN-gamma-positive cells by immunofluorescence microscopy and flow cytometry was noted. However, the latter has the advantage of a standardized control, is less labor consuming and is observer independent.

Adult↗
PubMed1977

Mepacrine, a tool for investigating the 5-hydroxytryptamine organelles of blood platelets by fluorescence microscopy.

In the blood platelets of various species exposed to mepacrine, the average number of green-yellow fluorescent granules (probably identical with the 5-hydroxytryptamine [5-HT] storage organelles) corresponded to that of flashes emitted by the platelets on prolonged irradiation with violet-blue light. In platelets of fawn-hooded rats the number of granules did not markedly differ from that of normal rat platelets, but the fluorescence intensity and the uptake of mepacrine in vitro showed a marked decrease and the flashes were less numerous. The heavy population of human platelets exhibited considerably more granular structures than the light population. The data suggest that (1) in normal, mepacrine-loaded platelets one flash corresponds to one 5-HT organelle and (2) mepacrine is a useful tool for investigating the number and function of the 5-HT organelles in live platelets and possibly for studying platelet age.

Animals↗
PubMedDate not supplied

Diagnosis of malaria by acridine orange fluorescent microscopy in an endemic area of venezuela.

Fluorescent (acridine orange) microscopical examination of capillary centrifuged blood (quantitative buffy coat [QBC] analysis) and Giemsa stained thick blood smears (GTS) were compared for diagnosis of malaria in blood specimens from adults living in malaria transmission areas of the States of Bolivar and Amazonas in southeastern and south Venezuela, respectively. Of a total of 198 GTS examined, 95 subjects (48%) showed parasitaemia. Among the 95 blood films with a positive GTS, 94 were judged positive by the QBC. However, positive QBC tubes were found in 29 out of 103 blood specimens with a negative GTS. Thus, relative to a GTS standard, the sensitivity and specificity of the QBC-test was 99.2% and 72%, respectively. Young trophozoites of Plasmodium vivax and P. falciparum could not be distinguished with certainty. It is confirmed that the QBC offers many advantages compared with the standard diagnosis of malaria parasites, specifically in the speed of staining and ease of interpretation. However, in places where P. falciparum and P. vivax occur, species and stage differentiation should be confirmed with the GTS.

Acridine Orange↗
PubMed1997

Cell type and spatial location dependence of cytoplasmic viscosity measured by time-resolved fluorescence microscopy.

Information on the cell type and spatial location dependence of cytoplasmic viscosity would be very useful in understanding some of the processes occurring in the cell. For this purpose, fluorescent dye kiton red (sulforhodamine B) was loaded into a variety of cells such as Swiss 3T3 fibroblasts, human mononuclear cells, Sarcoma-180 tumor cells, Chinese hamster ovary cells, plant cells from Digitalis lanata, stamen hair cells of Tradescantia, and guard mother cells of Allium cepa. Space-resolved measurements of cytoplasmic viscosity were carried out by using an experimental set-up wherein a picosecond laser system was coupled with an epifluorescence microscope. The spatial resolution of this set-up was approximately 1.0 micron, and reliable dynamic fluorescence measurements could be obtained from 10(2) to 10(3) fluorescent molecules. Fluorescence lifetime measurements showed that a large fraction (approximately 70%) of kiton red was in the free form. Fluorescence anisotropy decay of kiton red in cells was analyzed by a two population (free and bound) model. The microviscosity of cytoplasm was estimated from the anisotropy decay kinetics of the free probe. It was found that the cytoplasmic viscosity is dependent on both the cell type and spatial location within a cell. Furthermore, both the average value of viscosity and spatial variation within a cell were larger in the plant cells when compared to the animal cells. Model studies in various simpler systems have shown that the higher viscosity observed in some part of the cell could be due to either physical restriction and/or the presence of high concentrations of small solutes and macromolecules.

3T3 Cells↗
PubMed2005

Electron transfer reaction in a single protein molecule observed by total internal reflection fluorescence microscopy.

To observe an electron transfer (ET) process in a single protein molecule, we constructed a model system, Alexa-HCytb5, in which cytochrome b5 (Cytb5) is modified with a fluorescent probe, Alexa Fluor 647 dye. In this model system, intramolecular transfer of an electron from the Alexa dye to heme in Cytb5 is supposed to oxidize the probe and quench its fluorescence, and the ET reaction at the single-molecule level can be monitored as the intermittent change in the fluorescence intensity. Alexa-HCytb5 was fixed on the glass surface, and illumination of laser light by the total internal reflection resulted in blinking of the fluorescence from the single Alexa-HCytb5 molecule in the time scale of several hundred milliseconds. Each Alexa-HCytb5 molecule is characterized by its own rate constant of the blinking, corresponding to the ET rate constant at the single-molecule level, and its variation ranges between 1 and 10 s(-1). The current system thus enables us to visualize the ET reaction in the single protein molecule, and the protein ET reaction was found to be explained by the distribution of the rate constants. On the basis of the Marcus theory, we suggest that the origin of this rate distribution is the distance change associated with the structural fluctuation in the protein molecule.

Cytochromes b5↗
PubMed1991

Positive typing of Candida using fluorescent microscopy.

A pilot study was carried out to identify three species of candida using the 10 second staining technique with fluorescent dyes. Based on the morphology and the internal characteristics, a positive identification of each species was possible.

Candida↗
PubMed2001

Artefacts in restored images due to intensity loss in three-dimensional fluorescence microscopy.

Computational algorithms for three-dimensional deconvolution have proven successful in reducing blurring and improving the resolution of fluorescence microscopic images. However, discrepancies between the imaging conditions and the models on which such deconvolution algorithms are based may lead to artefacts and/or distortions in the images restored by application of the algorithms. In this paper, artefacts associated with a decrease of fluorescence intensity with time or slice in three-dimensional wide-field images are demonstrated using simulated images. Loss of intensity, whether due to photobleaching or other factors, leads to artefacts in the form of bands or stripes in the restored images. An empirical method for correcting the intensity losses in wide-field images has been implemented and used to correct biological images. This method is based on fitting a decreasing function to the slice intensity curve computed by summing all pixel values in each slice. The fitted curve is then used for the calculation of correction factors for each slice.

Algorithms↗