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At least 1,117 records · Page 62Linked to original sources
PubMed2002

Fibre-optic two-photon scanning fluorescence microscopy.

Two geometries of a novel two-photon fluorescence microscope incorporating single-mode fibre optics for the delivery of ultrashort-pulsed illumination to a remote sample are characterized. First, a 785 nm single-mode optical fibre is implemented in a scanning microscope, which demonstrates that an improvement in axial resolution is achieved due to the non-linear response of the fibre to intense ultrashort-pulsed light. Second, a 785 nm single-mode optical fibre coupler is adapted, in which case spectral broadening and blue shifting of the ultrashort-pulsed laser beam caused by the non-linear response of the fibre to ultrashort-pulsed illumination are experimentally characterized. An investigation into the impact of temporal broadening of the ultrashort-pulsed beam on the systems is also considered. The coupling efficiency of both geometries for various illumination wavelengths is also presented. The introduction of the fibre coupler to the system has significant advantages, including an improved optical sectioning effect and a reduction in the number of bulk optical components resulting in a low-cost, compact instrument. Sets of three-dimensional images of fluorescent polymer microspheres and biological material confirm these features.

Eukaryota↗
PubMed2003

Chemical heterogeneity in cell death: combined synchrotron IR and fluorescence microscopy studies of single apoptotic and necrotic cells.

The combination of synchrotron IR microspectroscopy and fluorescence microscopy has led to the identification of specific IR signatures of apoptosis and necrosis at a single cell level. Apoptosis was induced by treatment of Fas+ tumor cell lines with anti-Fas monoclonal antibodies. Detection of the early and late stages of apoptosis was performed using conjugated annexin V-fluorescein isothiocyanate (AV-FITC) and propidium iodide. Very early cellular changes were detected by IR before externalization of phosphatidylserine and AV-FITC labeling, and they were probably linked to DNA unwinding. The IR signals at 1044, 1177, and 1222 cm(-1), as well as an intensity variation in the CHx stretching region, are the main signature changes of early and late apoptosis, in line with the hypothesis of DNA fragmentation. The increased intensity of the CHx stretching bands of the lipids was observed only at an early stage of apoptosis. Changes in the relative intensity of CH3 and CH2 stretching accompany this increased intensity, suggesting changes in the relative amount and/or type of lipids concomitant with an increased lipid content. Finally, necrotic cells were characterized by marked changes in their chemical composition because several new vibrational features were observed.

Animals↗
PubMed2006

Effective elimination of laser interference fringing in fluorescence microscopy by spinning azimuthal incidence angle.

Laser illumination used in both conventional widefield epi-fluorescence as well as in total internal reflection fluorescence (TIRF) microscopy is subject to nonuniformities in intensity that obscure true image details. These intensity variations are interference fringes arising from coherent light scattering and diffraction at every surface in the laser light's optical path, including the lenses, mirrors, and coverslip. We present an inexpensive technique for effectively eliminating these interference fringes based upon introduction of the excitation laser beam by oblique through-the-objective incidence coupled with rapid azimuthal rotation of the plane of incidence. Although this rotation can be accomplished in several ways, a particularly simple method applicable to a free laser beam is to use an optical wedge, spun on a motor, which diverts the beam into a hollow cone of fixed angle. A system of lenses converts this collimated beam cone into a focused spot that traces a circle at the objective's back focal plane. Consequently, a collimated beam with fixed polar angle and spinning azimuthal angle illuminates the sample. If the wedge is spun rapidly, then the different interference patterns at every particular azimuthal incidence angle average out over a single camera exposure to produce an effectively uniform field of illumination.

Algorithms↗
PubMed2003

Quantitative analysis of cytoskeletal organization by digital fluorescent microscopy.

BACKGROUND: The cytoskeleton consists of complex arrays of fibers that play indispensable roles in cell structure and function. The cytoskeletal fibers are concertedly involved in numerous cellular processes, including cell adhesion, locomotion, intracellular transport, and cell division. The organization of the cytoskeleton was extensively studied, mainly by immunofluorescence microscopy, yet these studies were mostly qualitative, and a reliable quantitative approach for determining fiber structure and distribution is still missing. METHODS: In this study we developed algorithms for filament feature extraction, based on fluorescence microscopy. These algorithms are robust against blurring by slight defocus, high background, and noise, and are applicable to both fixed, immunolabeled cells and live cells expressing fluorescently tagged cytoskeletal proteins. The implemented FiberScore program is used in order to recognize, segment, and quantify various structural parameters of the cytoskeleton, including total fiber-associated fluorescence, as well as fiber length and orientation. Furthermore, these parameters can be determined for different cytoskeletal proteins in the same sample tagged with multiple-fluorescent labels, and the results can be correlated with other cellular parameters. RESULTS: FiberScore was used here for the quantification of simultaneous changes in microtubule and actin filaments induced by the microtubule-disrupting drug nocodazole. Actin filaments, which are reported to respond reciprocally to microtubule disruption, are found to be affected by both immediate and delayed signals. CONCLUSIONS: Analysis of the organization of fibers by the FiberScore algorithm allows quantification of the cytoskeletal signature of cells and offers reliable multiparametric functional assays for effects of drugs and other perturbations evaluated on a cell-by-cell basis.

3T3 Cells↗
PubMed1991

Acridine orange fluorescent microscopy and the detection of malaria in populations with low-density parasitemia.

Detection of low-density malaria parasites with Giemsa-stained thick smears (G-TS) requires time and experience and becomes impractical with high sample loads. Acridine orange fluorescent microscopy (AO/FM) of capillary centrifuged blood may offer an alternative technique. We compared AO/FM readings with G-TS in 290 specimens from asymptomatic people in Thai villages endemic for malaria. AO/FM specimens were prepared in modified capillary tubes coated with acridine orange (Quantitative Buffy Coat or "QBC tubes") and examined under a fluorescent microscope. Twenty-three (85.2%) of the 27 specimens found positive by G-TS had under 100 parasites/microliters blood (less than 35 parasites/200 microscopic fields). The overall AO/FM sensitivity was 78.9% [range: 66.7% (10/15)-86.7% (13/15)]. For Plasmodium falciparum, regardless of stages, the sensitivities varied from 66.7% (8/12) to 91.7% (11/12). AO/FM performed better for P. falciparum than for Plasmodium vivax and for asexual than for sexual stages of the parasite. However, the species- and stage-specific results must be interpreted with caution because of the small sample sizes and very low parasite densities involved. The test specificity was 96.6% [range: 95.6% (263/275)-97.1% (263/271)]. These levels of accuracy plus the known advantages of AO/FM suggest that the test, supplemented with G-TS to improve species and stage differentiation, is also useful for screening low-density parasitemias.

Acridine Orange↗
PubMed2006

Using quantitative fluorescence microscopy and FRET imaging to measure spatiotemporal signaling events in single living cells.

The mechanisms that mediate how migratory eukaryotic cells amplify a shallow, extracellular chemoattractant gradient into a steep intracellular gradient of signaling components to guide chemotaxis remains unknown. To unravel these mechanisms, it is essential to quantitatively measure the spatiotemporal patterns of chemoattractant gradients, the dynamic movement of intracellular signaling pathway molecules, and the localized activation of these molecules in single living cells. Recent developments in live-cell fluorescence microscopy have permitted direct visualization and quantitative measurement of signal transduction events with high temporal and spatial resolution. Here, we outline fluorescence imaging methods to simultaneously visualize and quantitatively measure spatiotemporal changes in chemoattractant concentration by using the fluorescent tracer dye Alexa 594. Next, we provide a method to correlate the dynamic changes in ligand to the spatiotemporal changes in the second messenger phosphatidylinositol 3,4,5-triphosphate (PIP3) along the inner surface of the plasma membrane in live cells. Finally, we describe a fluorescence resonance energy transfer (FRET) method to determine the extent of heterotrimeric G protein activation in single living cells in response to various chemoattractant fields.

Animals↗
PubMed2004

Viability of endolithic micro-organisms in rocks from the McMurdo Dry Valleys of Antarctica established by confocal and fluorescence microscopy.

The rocks of the McMurdo Dry Valleys desert in Antarctica harbour endolithic communities of micro-organisms such as lichens, fungi, cyanobacteria and bacteria. Establishing the physiological status and viability of these microbial colonies in their natural microhabitat has far-reaching implications for understanding the microbial ecology of the harsh environment of this polar desert. Here we describe the use of confocal microscopy and a specific fluorescent probe (FUN-1) to evaluate the metabolic activity of fungal cells. Application of confocal microscopy also served to identify living and dead bacteria or cyanobacteria using the fluorescent assay reagents Live/Dead SYTO 9 and propidium iodide or SYTOX Green, respectively. In addition, through the use of epifluorescence microscopy, live/dead bacteria and cyanobacteria could be detected by estimating fluorescence from their cell components provoked by simultaneously staining with nucleic acids stains such as DAPI and SYTOX Green.

Antarctic Regions↗
PubMed1976

[Study of the innervation of blood vessels of the digestive organs by fluorescence microscopy].

The blood vessels of the samll intestine and the gallbladder were shown to possess a great amount of adrenergic nerve fibres which, when penetrating the thickness of the wall of the above organs, become thinner and the distributed between the tissue structures of the organs as the thinnest monoaxonal network. The method of Falck--Hillarp--Krokhina was used. Among the vessel nerves there are perivascular nerves accompanying the vessels along their total legnth, juxtavascular and intramural nervous bundles of the sumpathetic nature detected by the fluorescent-microscopy method. Large arteries are disposed in a considerably thicker network of specifically fluorescing fibres than veins and small arteries.

Adrenergic Fibers↗
PubMed2004

Ultrasensitive confocal fluorescence microscopy of C-reactive protein interacting with FcgammaRIIa.

BACKGROUND: C-Reactive protein (CRP) is an acute phase protein with a suggested pathogenic role in cardiovascular disease. Previous reports proposed that the low-affinity IgG receptor FcgammaRIIa is the major receptor for CRP. However, these reports were met with criticism because the use of anti-CRP antibodies in the detection of CRP binding to FcgammaRIIa may have caused false-positive results. METHODS AND RESULTS: To resolve this controversy, we used ultrasensitive fluorescence microscopy to study the association, dissociation, and equilibrium of CRP binding to FcgammaRIIa. CRP indeed binds to FcgammaRIIa, with low association rates and dissociation rates. Anti-CRP antibodies markedly enhance binding, as is evident from the decrease of the equilibrium dissociation coefficient by 2 orders of magnitude. CONCLUSIONS: Our study demonstrates the virtues of single fluorophore labeling and highlights the pitfalls of immunolabeling in investigating CRP/Fc receptor interactions. Importantly, this article provides the first quantitative characterization of CRP binding to FcgammaRIIa and explains and reconciles the diverse and conflicting data presented in the literature.

Animals↗
PubMed2001

Thiazolidinedione toxicity to isolated hepatocytes revealed by coherent multiprobe fluorescence microscopy and correlated with multiparameter flow cytometry of peripheral leukocytes.

Thiazolidinediones (TZDs) are effective for the treatment of adult-onset insulin-resistant diabetes. Unfortunately, TZDs are associated with sporadic hepatic dysfunction that is not predictable from experimental animal studies. We investigated the response of isolated rat and human hepatocytes to various TZDs using biochemical assays, coherent multiprobe fluorescence microscopy and flow cytometric analyses. The results identified direct effects of TZD on mitochondria from live human and rodent hepatocytes. The multiprobe fluorescence assays showed disruption of mitochondrial activity as an initiating event followed by increased membrane permeability, calcium influx and nuclear condensation. Other TZD-related cellular effects were increased hepatic enzyme leakage, decreased reductive metabolism and cytoplasmic adenosine triphosphate depletion. Mitochondrial effects were similar in cryopreserved hepatocytes from diabetic or non-diabetic donors. Peripheral blood mononuclear cells (PBMCs) had baseline mitochondrial energetics and metabolism comparable with isolated hepatocytes. Mitochondrial effects in isolated hepatocytes were found in human PBMCs exposed to the TZDs. The relative potency of TZDs for causing hepatocyte and PBMC effects was troglitazone > pioglitazone > rosiglitazone. These studies clearly demonstrated that hepatic alterations in vitro are characteristic of TZDs, with only quantitative differences in subcellular organelle dysfunction. Monitoring mitochondrial function in isolated PBMCs may be beneficial in diabetics undergoing TZD therapy.

Adenosine Triphosphate↗
PubMed1957

Detection of gynecological cancer; use of fluorescence microscopy to show nucleic acids in malignant growth.

Early detection of malignant lesions of the cervix, a major problem in gynecology, has been made possible in more cases by the development of exfoliative cytology. Mass-screening programs have been impeded, however, by the demands on time and skill of the examiner as posed by conventional techniques.A new method in exfoliative cytology, using fluorescence microscopy, essentially reduces the time of processing as well as of scanning of specimens. Suspicious cells show flaming orange-red fluorescence of the cytoplasm on a black background, impressively distinct from normal cells and giving a warning signal to the examiner. This color reaction is based upon cytochemical changes-namely, the abundance of ribonucleic acid in vividly growing and especially malignant cells.Besides gynecological material, the method is applicable to other forms of malignant disease.

Cytodiagnosis↗
PubMed1991

Toxicity in vital fluorescence microscopy: effect of dimethylsulfoxide, rhodamine-123, and DiI-low density lipoprotein on fibroblast growth in vitro.

Fluorescence microscopy performed on living cells is a valuable technique for elucidating patterns of cell growth in vitro over artificial biomaterials such as vascular grafts, and for in vivo studies such as identification and treatment of atherosclerotic plaques. Two fluorescent dyes of particular value for vital fluorescence studies are Rhodamine-123 and 3,3'-dioctadecylindocarbocyanine-labeled low density lipoprotein (DiI-LDL). We examined the toxicity of these two dyes and of dimethylsulfoxide (DMSO), a solvent used in Rhodamine-123 studies, on the growth of MRC5 human fetal fibroblasts in monolayer culture. Two parameters of cell growth were quantitated: Cell number (a measure of proliferation), and cell area (a measure of cell spreading), based on microscopic images obtained at the start and end of a 48-h growth period after brief exposure (0.5 h) to test solutions. We found that the recommended solvent for solubilization of Rhodamine-123, DMSO, caused cessation of cell proliferation and actual reduction in the area covered by adherent fibroblasts at concentrations of as low as 0.1% (vol:vol). Rhodamine-123 made up from an aqueous stock solution modestly retarded proliferation and spreading, and there was no significant effect of DiI-LDL on these parameters over prolonged periods of exposure (up to 24 h) in culture. These results demonstrate that the most toxic substance for growing fibroblasts was the solvent DMSO. We conclude that both the solvent vehicle and fluorescent dye should be carefully examined for potential toxicity before such dyes are used for vital fluorescence studies of living cells.

Carbocyanines↗
PubMed2006

Filter cubes with built-in ultrabright light-emitting diodes as exchangeable excitation light sources in fluorescence microscopy.

The use of ultrabright light-emitting diodes as a potential substitute for conventional excitation light sources in fluorescence microscopy is demonstrated. We integrated ultrabright light-emitting diodes in the filter block of a conventional fluorescence microscope together with a collimating Fresnel lens, a holographic diffuser and emission filters. This setup enabled convenient changes between different excitation light sources and resulted in high excitation efficiencies. Quantitative comparison of image intensities of test samples revealed that light-emitting diodes yielded intensities in the range of a mercury arc lamp depending on the wavelength. The use of ultrabright light-emitting diodes also enabled luminescence lifetime imaging without the need for image intensification.

Journal Article↗
PubMed1994

Analysis by fluorescence microscopy of the development of compartment-specific gene expression during sporulation of Bacillus subtilis.

The use of a fluorogenic substrate, 5-octanoylaminofluorescein-di-beta-D-galactopyranoside, for beta-galactosidase has made it possible to visualize enzyme activity in individual cells of sporulating populations of Bacillus subtilis by fluorescence microscopy. lacZ fusions to different sporulation-associated genes have been used to investigate the cell compartmentalization of gene expression during sporulation. A strain with a lacZ fusion to sspA, a gene which is transcribed by E-sigma G at a late stage of sporulation, displayed predominantly compartment-specific fluorescence. Expression of the early-expressed spoIIA locus, which includes the structural gene for sigma F, was seen not to be compartmentalized. Populations of strains with lacZ fusions to gpr and dacF, genes which are transcribed by E-sigma F at intermediate stages of sporulation, included some organisms showing uncompartmentalized fluorescence and others showing compartment-specific fluorescence; the proportion showing compartment-specific fluorescence increased in samples taken later in sporulation. Several possible explanations of the results obtained with gpr and dacF are considered. A plausible interpretation is that sigma F activity is initially not compartmentalized and becomes compartmentalized as sporulation progresses. The progression to compartmentalization does not require the activities of the sporulation-specific factor sigma E or sigma G but may require some product of sigma F activity.

Adhesins, Bacterial↗
PubMed1997

Intravital fluorescence microscopy: impact of light-induced phototoxicity on adhesion of fluorescently labeled leukocytes.

Alterations in leukocyte/endothelium interaction due to phototoxic effects of the fluorescent dyes acridine orange (AO) and rhodamine 6G (Rh6G) were studied by intravital microscopy using the dorsal skinfold model in awake Syrian golden hamsters. AO (0.5 mg/kg/min; constant IV infusion) and Rh6G (0.1 micromol/kg; bolus IV) were administered via an indwelling venous catheter. Five to seven arterioles (35-55 microm) and postcapillary venules (30-65 microm) were investigated in each animal. Vessels were exposed four times for 30 sec to continuous light of the appropriate excitation wavelength with a 10-15-min time interval between exposures. Animals were randomly assigned to five experimental groups (five distinct light energy levels). AO and Rh6G induced leukocyte rolling/sticking in postcapillary venules and arterioles when exposed to high light energy levels. AO, but not Rh6G, induced arteriolar vasospasm when exposed to high light energies. The potential phototoxic effect of AO and Rh6G is demonstrated, as assessed by the stimulation of leukocyte-endothelium interaction and arteriolar vasospasm in vivo. This study underscores the necessity to optimize microscopic set-ups for intravital microscopy, to reduce the excitation light energy level significantly, and to perform stringent control experiments, ruling out an artificial phototoxicity-induced stimulation of leukocyte adhesion.

Acridine Orange↗