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 1,567 records · Page 87Linked to original sources
PubMed2006

Molecular analysis of mitotic chromosome condensation using a quantitative time-resolved fluorescence microscopy assay.

Chromosomes condense during mitotic entry to facilitate their segregation. Condensation is typically assayed in fixed preparations, limiting analysis of contributing factors. Here, we describe a quantitative method to monitor condensation kinetics in living cells expressing GFP fused to a core histone. We demonstrate the utility of this method by using it to analyze the molecular requirements for the condensation of holocentric chromosomes during the first division of the Caenorhabditis elegans embryo. In control embryos, the fluorescence intensity distribution for nuclear GFP:histone changes during two distinct time intervals separated by a plateau phase. During the first interval, primary condensation converts diffuse chromatin into discrete linear chromosomes. After the plateau, secondary condensation compacts the curvilinear chromosomes to form shorter bar-shaped structures. We quantitatively compared the consequences on this characteristic profile of depleting the condensin complex, the mitosis-specific histone H3 kinase Aurora B, the centromeric histone CENP-A, and CENP-C, a conserved protein required for kinetochore assembly. Both condensin and CENP-A play critical but distinct roles in primary condensation. In contrast, depletion of CENP-C slows but does not prevent primary condensation. Finally, Aurora B inhibition has no effect on primary condensation, but slightly delays secondary condensation. These results provide insights into the process of condensation, help resolve apparent contradictions from prior studies, and indicate that CENP-A chromatin has an intrinsic role in the condensation of holocentric chromosomes that is independent of its requirement for kinetochore assembly.

Animals↗
PubMed2004

Mapping ALA-induced PPIX fluorescence in normal brain and brain tumour using confocal fluorescence microscopy.

We have mapped the microscopic distribution of aminolevulinic acid (ALA) induced protoporphyrin IX (PPIX) in normal rabbit brain and in brain tumour at various time intervals after i.v. injection of ALA. In normal brain, regions without a blood-brain barrier (BBB) such as the choroid plexus and meninges and also structures associated with the CSF synthesized the highest amounts of PPIX. We also detected ALA in the CSF within 2 h after ALA administration when the serum concentration was high. Our fluorescence studies have shown that ALA is able to cross an intact BBB and be metabolized in regions with a BBB. This is evident in regions of the brain such as the hippocampus, thalamus, cerebellar folia and brain stem that are remote from the meninges or choroid plexus. However, the relative peak PPIX fluorescence in these regions are much lower than in regions without a BBB. Our imaging studies have shown that neurons in different regions of normal brain with an intact BBB exhibit varying capacities to metabolize ALA. The grey matter regions of the cerebrum and cerebellum also showed higher PPIX fluorescence compared to white matter regions in these same structures. Our study showed that tumour tissue had the highest PPIX fluorescence compared to normal contralateral cerebral cortex. Maximum selectivity was achieved between tumour and normal tissue at 24 h. PPIX fluorescence in normal blood vessels appear to be mainly due to metabolism of internalized ALA from serum and not due to high PPIX levels. In conclusion, ALA does cross the BBB and all regions of the brain with the exception of the hypothalamus, exhibit a dynamic synthesis of PPIX from ALA. Increased PPIX accumulation in tumour and inflamed brain tissue in comparison to normal brain tissue suggest the feasibility of selective photosensitization of such tissue.

Aminolevulinic Acid↗
PubMed1989

[Fluorescence microscopy detection of mycopathogens using Rylux BSU].

Rylux BSU (Synthesia, Pardubice--Semtín), a derivative of diaminostilbendisulphonic acid, has a good affinity to chitin of fungal cell walls as well as to cellulose of algae. A histologic method using this compound for detection of fungi in dermatology is presented accompanied with results of a quick diagnostic procedure. Pathogenic fungi were stained with a 2 per cent solution of Rylux BSU in phosphate buffer (pH 7.3) or in a 2 per cent aqueous solution of NaOH with an admixture of 10 per cent dimethylsulphoxide. A yellow green fluorescence of the cell walls of fungi (tested on 52 species) and algae (2 species) was evoked by incident blue light (max. filter transmission for 405 nm) combined with a barrier filter (max. 578 nm). Fluorescence was not fading and remained stable in Canada balsam. Moreover, the method could be applied on sections previously stained (HE, Weigert, Giemsa, KOH-preparations) because of more information offered by a combined procedure.

Benzenesulfonates↗
PubMed2003

Optical bioimaging: from living tissue to a single molecule: calcium imaging in blood vessel in situ employing two-photon excitation fluorescence microscopy.

Recent developments in optoelectronics permit real-time Ca(2+) imaging of thin planes within cells utilizing laser scanning confocal microscopy (LSCM). However, a major complication associated with this imaging system involves increased phototoxicity with improved spatiotemporal resolution. Two-photon excitation microscopy (TPEM) helps to minimize phototoxicity due to the restriction of this technique to the volume proximal to the geometric focus of the light. In this study, the capability of Ca(2+) imaging was investigated employing recently developed real-time TPEM, RTS2000MP (Bio-Rad, Tokyo) with a mode-locked Ti-sapphire laser. Z-axis resolution of RTS2000MP with high NA objectives defined as full-width at half maximum (FWHM) with a 0.5-microm fluorescent bead provided values nearly identical to those obtained with LSCM at a small pinhole (0.2 mm) (approximately 0.6 microm). When serial sectioning of 21 sequential images at 0.3-microm intervals in cultured endothelial cells loaded with calcein and tetramethyl-rhodamine methylester were performed with TPEM, the z-axis resolution was higher than that observed with LSCM; moreover, the photobleaching rate was significantly lower than that obtained with LSCM. Maximum fluorescence intensities were detected at 780 nm in excitation spectra of fluo-3 and fluo-4 Ca(2+)-sensitive probes with TPEM. Fluorescence images in mouse arterial endothelial cells loaded with fluo-4 could be clearly visualized by TPEM in situ. Application of acetylcholine caused oscillatory increase in [Ca(2+)](i) of endothelial cells; subsequently, relaxation along the major axis of smooth muscle cells was evident. Furthermore, consecutive long-lasting experiments could be repeated with identical response in the same microscopic field. In conclusion, fluorescence imaging employing TPEM is useful for Ca(2+) imaging in blood vessels in situ.

Animals↗
PubMed2005

Simultaneous time- and wavelength-resolved fluorescence microscopy of single molecules.

Single fluorophores and single-pair fluorescence resonance energy transfer were studied with a new confocal fluorescence microscope that allows, for the first time, the wavelength and emission time of each detected photon to be simultaneously measured with single molecule sensitivity. In this apparatus, the photons collected from the sample are imaged through a dispersive optical system onto a time and position sensitive photon detector. For each detected photon the detection system records its wavelength, its emission time relative to the excitation pulse, and its absolute emission time. A histogram over many photons can generate a full fluorescence spectrum and correlated decay plot for a single molecule for any time interval. At the single molecule level, this approach makes possible entirely new types of temporal and spectral correlation spectroscopies. This paper presents our initial results on simultaneous time- and wavelength-resolved fluorescence measurements of single rhodamine 6G (R6G), tetramethylrhodamine (TMR), and Cy3 molecules embedded in thin films of poly(methyl methacrylate) (PMMA), and of single-pair fluorescence resonance energy transfer between two Alexa fluorophores spaced apart by a short polyproline peptide.

Journal Article↗
PubMed1991

Kupffer cell-mediated oxidative stress on colon cancer cell line visualized by digital imaging fluorescence microscopy.

Temporal and spatial changes of lipid peroxides in a cultured colon cancer cell line, Colo-205 cells, were investigated after culturing with Kupffer cells by using 2',7'-dichlorofluorescein diacetate and a digital imaging processor equipped with an inverted microscope. By this method, we successfully visualized the alteration of lipid peroxides in the individual cancer cell. Without any prior activation, Kupffer cells isolated from an intact rat liver caused rapid increase in the intensity of dichlorofluorescein in tumor cells in a time-dependent manner. The increase of the fluorescent intensity was significantly attenuated by pretreatment with superoxide dismutase. In ex vivo study using isolated perfused rat liver, dichlorofluorescein-preloaded cancer cells, which were transportally injected, were found to adhere to hepatic sinusoids and then to enhance their fluorescence. The present study suggested that the resident Kupffer cell-derived oxidative stress participates in the cytotoxic process against cancer cells by inducing intracellular lipid peroxidation. It may be sustained that Kupffer cells play a role in the host defence mechanisms against the liver metastasis of colon cancer cells.

Animals↗
PubMed1994

Algorithms for automated characterization of cell populations in thick specimens from 3-D confocal fluorescence microscopy data.

Methods are presented for the automated, quantitative and three-dimensional (3-D) analysis of cell populations in thick, essentially intact tissue sections while maintaining intercell spatial relationships. This analysis replaces current manual methods which are tedious and subjective. The thick sample is imaged in three dimensions using a confocal scanning laser microscope. The stack of optical slices is processed by a 3-D segmentation algorithm that separates touching and overlapping structures using localization constraints. Adaptive data reduction is used to achieve computational efficiency. A hierarchical cluster analysis algorithm is used automatically to characterize the cell population by a variety of cell features. It allows automatic detection and characterization of patterns such as the 3-D spatial clustering of cells, and the relative distributions of cells of various sizes. It also permits the detection of structures that are much smaller, larger, brighter, darker, or differently shaped than the rest of the population. The overall method is demonstrated for a set of rat brain tissue sections that were labelled for tyrosine hydroxylase using fluorescein-conjugated antibodies. The automated system was verified by comparison with computer-assisted manual counts from the same image fields.

Algorithms↗
PubMed1993

Measurement of microhemodynamics in the ventilated rabbit lung by intravital fluorescence microscopy.

Pulmonary microhemodynamic parameters were directly measured along with systemic pressures and cardiac output in the ventilated rabbit lung. Subpleural arterioles and venules ranging from 10 to 35 microns luminal diameter were investigated under zone 2 conditions, i.e., during inspiratory plateau at an airway pressure of 8 mmHg. Mean arteriolar and venular diameters (24.6 +/- 3.3 and 21.9 +/- 3.6 microns, respectively), mean red blood cell (RBC) fluxes (1,549 +/- 501 and 1,257 +/- 600 cells/s), and mean RBC velocities (0.79 +/- 0.21 and 0.82 +/- 0.21 mm/s) were measured using a fluorescence video-microscopic technique. Calculated microhematocrit (Hct mu) was below systemic values (Hctsys) (Hct mu/Hctsys: arterioles, 0.75 +/- 0.12; venules, 0.67 +/- 0.08). The mean capillary transit time of RBC was 0.47 +/- 0.16 s over a mean arteriovenous distance of 173 +/- 70 m. Significant correlations were demonstrated between microhemodynamic parameters. A correlation among cardiac output, pulmonary arterial pressure, and RBC velocity demonstrates the connection between macro- and microhemodynamics in the rabbit lung. In conclusion, the present model is the first one enabling the measurements of the principal circulatory determinants for gas exchange, i.e., microvascular blood flow, Hct mu, and capillary transit time of RBCs in the ventilated rabbit lung under simultaneous macrocirculatory control.

Animals↗
PubMed1995

[Visualization of keratocytes in the human cornea with fluorescence microscopy].

There is little information about the state of human keratocytes in longterm cornea storage and the influence of these cells on the survival of the corneal transplant after keratoplasty. Up to now stromal keratocytes have not been taken into consideration in regard to the culture conditions in cornea banking. Therefore, a method was used, first described by Poole et al., who stained porcine corneas using the Live/Dead-Assay (Molecular Probes Inc.). After staining porcine and human corneal slices with the fluorogenic substance calcein AM and the fluorochrome ethidium homodimer, we examined the fluorescing cells to acquire more knowledge about the density, morphology and vitality of human keratocytes. Living cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually non-fluorescent cell-permeant calcein AM to the intensely fluorescent calcein, producing an intense, uniform, green fluorescence in living cells. Ethidium homodimer enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence on binding nucleic acids, thereby producing a bright red fluorescence in dead cells. The examinations were performed using a conventional fluorescence microscope and a confocal slit scanning microscope. Like in the porcine corneas, we found three keratocyte subpopulations in the human corneas, but there were some differences in morphology and patterns of staining between the two species in the central and posterior stroma. After long periods of cornea storage we found signs of cell degeneration like loss of cytoplasmic processes and an increasing number of dead cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

High-speed, random-access fluorescence microscopy: II. Fast quantitative measurements with voltage-sensitive dyes.

An improved method for making fast quantitative determinations of membrane potential with voltage-sensitive dyes is presented. This method incorporates a high-speed, random-access, laser-scanning scheme (Bullen et al., 1997. Biophys. J. 73:477-491) with simultaneous detection at two emission wavelengths. The basis of this ratiometric approach is the voltage-dependent shift in the emission spectrum of the voltage-sensitive dye di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS). Optical measurements are made at two emission wavelengths, using secondary dichroic beamsplitting and dual photodetectors (<570 nm and >570 nm). Calibration of the ratiometric measurements between signals at these wavelengths was achieved using simultaneous optical and patch-clamp measurements from adjacent points. Data demonstrating the linearity, precision, and accuracy of this technique are presented. Records obtained with this method exhibited a voltage resolution of approximately 5 mV, without any need for temporal or spatial averaging. Ratiometric recordings of action potentials from isolated hippocampal neurons are used to illustrate the usefulness of this approach. This method is unique in that it is the first to allow quantitative determination of dynamic membrane potential changes in a manner optimized for both high spatiotemporal resolution (2 micrometers and <0.5 ms) and voltage discrimination.

Action Potentials↗
PubMed1984

Demonstration of beta-adrenergic receptors in rat bronchiolar epithelial cells employing 9-amino-acridyl propranolol fluorescent microscopy.

Prior work has provided ultrastructural evidence that beta-adrenergic agonists stimulate secretion by nonciliated bronchiolar epithelial (Clara) cells of the rat (J Clin Invest 67:345, 1981). However, since the lung is a multicellular organ it is not clear if the beta-agonists act directly on the Clara cell. The absence in Clara cells of beta-adrenergic receptors would indicate an indirect action of the beta-adrenergic agonists. In the present study, we used 9-amino-acridyl propranolol in an attempt to determine if beta-adrenergic receptors are present in rat bronchiolar Clara cells. Discrete, intense yellow fluorescent dots were identified microscopically in ciliated and in Clara cells of the rat. This anatomical localization of beta-adrenergic receptors supports the notion that beta-adrenergic agonists stimulate secretion by acting directly on Clara cells.

Animals↗
PubMed2004

Imaging coronary artery microstructure using second-harmonic and two-photon fluorescence microscopy.

The microstructural basis for the mechanical properties of blood vessels has not been directly determined because of the lack of a nondestructive method that yields a three-dimensional view of these vascular wall constituents. Here, we demonstrate that multiphoton microscopy can be used to visualize the microstructural basis of blood vessel mechanical properties, by combining mechanical testing (distension) of excised porcine coronary arteries with simultaneous two-photon excited fluorescence and second-harmonic generation microscopy. Our results show that second-harmonic generation signals derived from collagen can be spectrally isolated from elastin and smooth muscle cell two-photon fluorescence. Two-photon fluorescence signals can be further characterized by emission maxima at 495 nm and 520 nm, corresponding to elastin and cellular contributions, respectively. Two-dimensional reconstructions of spectrally fused images permit high-resolution visualization of collagen and elastin fibrils and smooth muscle cells from intima to adventitia. These structural features are confirmed by coregistration of multiphoton microscopy images with conventional histology. Significant changes in mean fibril thickness and overall wall dimension were observed when comparing no load (zero transmural pressure) and zero-stress conditions to 30 and 180 mmHg distension pressures. Overall, these data suggest that multiphoton microscopy is a highly sensitive and promising technique for studying the morphometric properties of the microstructure of the blood vessel wall.

Animals↗
PubMed2002

In vivo FM: using conventional fluorescence microscopy to monitor retinal neuronal death in vivo.

Post-traumatic death of mature retinal neurons occurs in glaucoma and after optic nerve injury. The death is a dynamic process that can be fully analyzed with methods that monitor changes over time. We have coupled the development of retrogradely transportable fluorescent dyes with modification of conventional epifluorescence microscopy to manipulate and visualize rat retinal neurons in vivo. The method is a relatively new concept and has potential for the monitoring of retinal conditions, such as glaucoma or optic nerve transection, and for evaluation of neuroprotective strategies in the near future.

Animals↗
PubMed1996

Effect of fixatives on calf thymocytes chromatin as analyzed by 3D high-resolution fluorescence microscopy.

Two common fixatives--glutaraldehyde and ethanol/acetic acid mixture--were studied to understand their effects on DNA distribution inside the cell nucleus. Native calf thymocytes were analyzed by using a DNA selective fluorescent dye (DAPI) and computational optical sectioning microscopy on isolated cells before and after fixation. In order to estimate quantitatively the intranuclear DNA distribution, the stained calf thymocytes images were processed by removing the out-of-focus contributions present in each optical section. Although preliminary, the results show that within individual nuclei the frequency distribution of the fluorescence intensity appears significantly and differentially altered by the two fixatives. Namely with respect to the native unfixed preparation, the ethanol/acetic acid causes the complete disappearance of the higher intensity pixels, whereas glutaraldehyde fixation can be associated with the appearance of new ones of an even higher intensity. The quantitative analysis of the processed images allowed us to reconstruct the topological distribution of DNA inside the nucleus and to correlate the data with the results obtained by differential scanning calorimetry on similar samples.

Acetates↗
PubMed2004

Measurement of SOS expression in individual Escherichia coli K-12 cells using fluorescence microscopy.

Many recombination, DNA repair and DNA replication mutants have high basal levels of SOS expression as determined by a sulAp-lacZ reporter gene system on a population of cells. Two opposing models to explain how the SOS expression is distributed in these cells are: (i) the 'Uniform Expression Model (UEM)' where expression is evenly distributed in all cells or (ii) the 'Two Population Model (TPM)' where some cells are highly induced while others are not at all. To distinguish between these two models, a method to quantify SOS expression in individual bacterial cells was developed by fusing an SOS promoter (sulAp) to the green fluorescent protein (gfp) reporter gene and inserting it at attlambda on the Escherichia coli chromosome. It is shown that the fluorescence in sulAp-gfp cells is regulated by RecA and LexA. This system was then used to distinguish between the two models for several mutants. The patterns displayed by priA, dnaT, recG, uvrD, dam, ftsK, rnhA, polA and xerC mutants were explained best by the TPM while only lexA (def), lexA3 (ind-) and recA defective mutants were explained best by the UEM. These results are discussed in a context of how the processes of DNA replication and recombination may affect cells in a population differentially.

Adenosine Triphosphatases↗
PubMed1989

Non-invasive intravital fluorescent microscopy of the hamster gingiva.

Several attempts have been made to visualize the gingival microcirculation of small animals. Most of these attempts have required surgical procedures without control of the local tissue environment. In our current study of the hamster gingiva, a tissue bath was designed to allow microcirculatory observations of the periodontium of the superior incisors without the need for invasive surgical procedures and with controlled pH and temperature of the bathing solution. Dextrans (m.w. = 70,000 and m.w. = 150,000), bovine serum albumin (mw = 66,210) or a globulin fraction (mw = greater than 450,000 less than 825,000) were conjugated to fluorescein isothiocyanate and injected into the animal. Fluorescent images, produced by epi-illumination, were analyzed for the light intensity of the interstitium. There was a large and rapid increase in light intensity (leakage of the macromolecules) near the venules while capillaries of the gingival margin developed interstitial fluorescence at a slower rate and with a lower maximal effect. Molecular weight, shape or charge of the macromolecular or age of the animal did not affect the results. This new preparation could be used to study the contribution of the microcirculation to gingivitis, the role of gingivitis in the development of periodontal disease, or the effects of drugs and hormones on the microcirculation of the gingiva.

Animals↗
PubMed2004

The use of optical parametric oscillator for harmonic generation and two-photon UV fluorescence microscopy.

Ultrafast lasers have found increasing use in scanning optical microscopy due to their very high peak power in generating multiphoton excitations. A mode-locked Ti:sapphire laser is often employed for such purposes. Together with a synchronously pumped optical parametric oscillator (OPO), the spectral range available can be extended to 1,050-1,300 nm. This broader range available greatly facilitates the excitation of second harmonic generation (SHG) and third harmonic generation (THG) due to better satisfaction of phase matching condition that is achieved with a longer excitation wavelength. Dental sections are then investigated with the contrasts from harmonic generation. In addition, through intra-cavity doubling wavelengths from 525-650 nm are made available for effective two-photon (2-p) excitation with the equivalent photon energy in the UVB range (290-320 nm) and beyond. This new capacity allows UV (auto-) fluorescence excitation and imaging, for example, from some amino acids, such as tyrosine, tryptophan, and glycine.

Dentin↗
PubMed2002

Measuring tubulin content in Toxoplasma gondii: a comparison of laser-scanning confocal and wide-field fluorescence microscopy.

Toxoplasma gondii is an intracellular parasite that proliferates within most nucleated cells, an important human pathogen, and a model for the study of human and veterinary parasitic infections. We used a stable yellow fluorescent protein-alpha-tubulin transgenic line to determine the structure of the microtubule cytoskeleton in T. gondii. Imaging of living yellow fluorescent protein-alpha-tubulin parasites by laser-scanning confocal microscopy (LSCM) failed to resolve the 22 subpellicular microtubules characteristic of the parasite cytoskeleton. To understand this result, we analyzed sources of noise in the LSCM and identified illumination fluctuations on time scales from microseconds to hours that introduce significant amounts of noise. We confirmed that weakly fluorescent structures could not be imaged in LSCM by using fluorescent bead standards. By contrast, wide-field microscopy (WFM) did visualize weak fluorescent standards and the individual microtubules of the parasite cytoskeleton. We therefore measured the fluorescence per unit length of microtubule by using WFM and used this information to estimate the tubulin content of the conoid (a structure important for T. gondii infection) and in the mitotic spindle pole. The conoid contains sufficient tubulin for approximately 10 microtubule segments of 0.5-microm length, indicating that tubulin forms the structural core of the organelle. We also show that the T. gondii mitotic spindle contains approximately 1 microtubule per chromosome. This analysis expands the understanding of structures used for invasion and intracellular proliferation by an important human pathogen and shows the advantage of WFM combined with image deconvolution over LSCM for quantitative studies of weakly fluorescent structures in moderately thin living cells.

Algorithms↗