[Microanalysis of fluorescence--spectrometry of fluorescent microscopy].
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Candida↗
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Results for “Fluorescence Microscopy”
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The fate of autoreactive, GFP+ T cells in rat models of uveitis analyzed by intravital fluorescence microscopy and FACS.
Experimental autoimmune uveitis (EAU) is an inflammatory disease of the immune privileged inner eye, mediated by CD4(+) Th1 cells specific for retinal autoantigens. To elucidate the fate of the T cells in the eye we adoptively transferred green fluorescent protein-positive (GFP(+)) T cells with specificity for R14, a peptide from interphotoreceptor retinoid-binding protein (IRBP) or OVA as foreign control antigen to naive Lewis rats. We also used the model of immunogenic uveitis, an inflammatory eye disease induced by intraocular injection of soluble OVA 1 day post transfer of OVA-specific GFP(+) cells. We investigated the timing of ocular T cell infiltration and their immunological activation state by intravital fluorescence microscopy (IVFM) of the iris until onset of intraocular inflammation. Within 30 min of injection, GFP(+) cells invaded the iris tissue, irrespective of their antigen specificity, whereas intraocular inflammation was only observed 3 days later, if cells recognized their respective antigen (R14-specific cells in EAU, OVA-specific cells in immunogenic uveitis). Using FACS analysis we found that activation markers were upregulated only on cells from uveitic eyes, but not from other sources, suggesting that intraocularly presented specific antigen is a prerequisite for T cell reactivation and subsequent recruitment of inflammatory cells.
Animals↗
FLUORESCENCE MICROSCOPY AND AUTORADIOGRAPHY OF COLCHICINE-INDUCED MICRONUCLEATED CELLS.
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Acridines↗
Intravital fluorescence microscopy for the study of blood-brain-barrier function.
Vascular diameters and blood-brain-barrier (BBB) function were investigated in the parietal cortex of cats using an open skull window technique and intravital fluorescence microscopy. The cortical surface was superfused with artificial cerebrospinal fluid containing: bradykinin (BK), Na+-arachidonate (AA), or xanthine-oxidase (XO). Na+-fluorescein (MW: 376), fluorescein-isothiocyanate-labelled (FITC) albumin (MW: 67 000), or FITC-dextran (MW: 19 400-62 000) were given intravenously as blood-brain-barrier indicators of different molecular size. In control experiments, the effect of continuous exposure of the preparation to the light source used for fluorescence excitation was studied. Dependent on the molecular size of the tracer and light intensity, continuous light exposure led to extravasation and disturbances of the microcirculation in small veins. When Na+-fluorescein was employed as barrier indicator at a magnification of X 40, at least 55 min of continuous illumination were required to induce extravasation. Brief, i.e. 0.5-15 s and discontinuous illumination for taking microphotographs amounting to less than 20 min in total did not induce extravasation in control experiments of 3.5 h. Opening of the blood-brain-barrier was studied during superfusion with a hypertonic solution (2000 mOsmol 1(-1)). The results obtained with bradykinin, Na+-arachidonate, or xanthine-oxidase indicate that opening of the barrier can occur independently from a corresponding vasodilating reaction. BK, or AA led to initial venular leakage as a result of an increase of selective, or global barrier permeability. On the other hand, XO did not induce extravasation, although cerebral vessels were markedly dilated. Taken together, the experimental model presented is suitable to simultaneously analyze dynamic changes of the permeability of cerebral vessels in-vivo with excellent spatial resolution and of the cerebral vasomotor behaviour under physiological and pathological conditions.
Animals↗
Analysis of transferrin recycling in mitotic and interphase HeLa cells by quantitative fluorescence microscopy.
Recent findings suggest that membrane vesicle transport during mitosis may be generally inhibited. To test this, we examined the kinetics of uptake and exocytosis of RITC-transferrin in mitotic and interphase HeLa cells. We used quantitative image-intensification fluorescence microscopy to analyze the content of ligands in single cells. This technique was validated by comparison of 3H or RITC-transferrin release from interphase cells determined by microscopy or radiometry. Both methods gave a t1/2 of release of 5-6 min. The uptake of RITC-transferrin was depressed in mitotics. More importantly, we monitored the exocytosis of label during mitosis. Labeled mitotics were obtained by the progression of interphase cells into mitosis during a 50 min incubation with RITC-transferrin. After 30 min chase with unlabeled transferrin, the intensities of interphase cells approached background, whereas those of mitotic cells remained nearly constant. Thus both exocytosis and endocytosis of transferrin were exocytosis and endocytosis of transferrin were blocked during mitosis.
Fluorescent Dyes↗
Location of P-II and AlPCS4 in human tumor LOX in vitro and in vivo by means of computer-enhanced video fluorescence microscopy.
The patterns of in vitro intracellular and in vivo intratumoral localization of Photofrin II (P-II) and aluminum phthalocyanine tetrasulfonate (AlPCS4) in human melanoma LOX were studied by means of computer-enhanced video fluorescence microscopy (CEVFM). The hydrophobic drug P-II localized diffusely in the perinuclear fraction of the cytoplasm of the LOX cells cultivated in vitro. Light exposure did not result in any observable change in the localization pattern. The hydrophilic dye AlPCS4 was distributed as granular and grain patterns in the cytoplasm before light exposure, in exactly the same pattern as that of acridine orange incubated in the same cells, which is known to emit red fluorescence from lysosomes, thus indicating that AlPCS4 was also primarily localized in the lysosomes of the LOX cells. After light exposure the distribution of the intracellular AlPCS4 fluorescence was altered and the intensity increased. In vivo, P-II had a combined cellular localization pattern (i.e. a strongly cytoplasmic membrane-localizing pattern and a weakly intracellular distribution pattern) and an extracellular distribution pattern in the tumor tissue, while the AlPCS4 fluorescence was seen mainly in the stroma of the tumor. The total fluorescence intensity of P-II and AlPCS4 in the LOX tumor tissue at different times after injection was quantitatively determined by means of CEVFM.
Animals↗
Fluorescence microscopy in vaginal cytology.
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Cytodiagnosis↗
Nanometer-localized multiple single-molecule fluorescence microscopy.
Fitting the image of a single molecule to the point spread function of an optical system greatly improves the precision with which single molecules can be located. Centroid localization with nanometer precision has been achieved when a sufficient number of photons are collected. However, if multiple single molecules reside within a diffraction-limited spot, this localization approach does not work. This paper demonstrates nanometer-localized multiple single-molecule (NALMS) fluorescence microscopy by using both centroid localization and photobleaching of the single fluorophores. Short duplex DNA strands are used as nanoscale "rulers" to validate the NALMS microscopy approach. Nanometer accuracy is demonstrated for two to five single molecules within a diffraction-limited area. NALMS microscopy will greatly facilitate single-molecule study of biological systems because it covers the gap between fluorescence resonance energy transfer-based (<10 nm) and diffraction-limited microscopy (>100 nm) measurements of the distance between two fluorophores. Application of NALMS microscopy to DNA mapping with <10-nm (i.e., 30-base) resolution is demonstrated.
DNA↗
Immunodetection and fluorescent microscopy of transgenically expressed hordeivirus TGBp3 movement protein reveals its association with endoplasmic reticulum elements in close proximity to plasmodesmata.
The subcellular localization of the hydrophobic TGBp3 protein of Poa semilatent virus (PSLV, genus Hordeivirus) was studied in transgenic plants using fluorescent microscopy to detect green fluorescent protein (GFP)-tagged protein and immunodetection with monoclonal antibodies (mAbs) raised against the GFP-based fusion expressed in E. coli. In Western blot analysis, mAbs efficiently recognized the wild-type and GFP-fused PSLV TGBp3 proteins expressed in transgenic Nicotiana benthamiana, but failed to detect TGBp3 in hordeivirus-infected plants. It was found that PSLV TGBp3 and GFP-TGBp3 had a tendency to form large protein complexes of an unknown nature. Fractionation studies revealed that TGBp3 represented an integral membrane protein and probably co-localized with an endoplasmic reticulum-derived domain. Microscopy of epidermal cells in transgenic plants demonstrated that GFP-TGBp3 localized to cell wall-associated punctate bodies, which often formed pairs of opposing discrete structures that co-localized with callose, indicating their association with the plasmodesmata-enriched cell wall fields. After mannitol-induced plasmolysis of the leaf epidermal cells in the transgenic plants, TGBp3 appeared within the cytoplasm and not at cell walls. Although TGBp3-induced bodies were normally static, most of them became motile after plasmolysis and displayed stochastic motion in the cytoplasm.
Blotting, Western↗
Simultaneous localization of six antigens in single sections of transgenic mouse intestine using a combination of light and fluorescence microscopy.
To study the geographic differentiation of the intestinal epithelium and to understand the complex lineage relationships of its cell populations, it is often necessary to visualize the protein products of multiple genes in sections prepared from different positions along the duodenal-to-colonic and/or crypt-to-villus axes. Multilabel fluorescence or brightfield immunohistochemical techniques have previously been used for this purpose. However, the number of antigens that can be identified on single sections is limited in fluorescence microscopy by the number of fluorophores with non-overlapping absorption and emission characteristics, in brightfield microscopy by the number of visually distinguishable chromogens, and in both methods by the availability of primary antisera raised in multiple species. We have now used a combination of light and fluorescence microscopic techniques to increase the number of antigens that can be detected in a single section to six. Sections were sequentially stained using immunogold with silver intensification, peroxidase-antiperoxidase with diaminobenzidine chromogen, and peroxidase-anti-peroxidase with alpha-naphthol/basic dye as chromogen, followed by simultaneous fluorescent detection with fluorescein, 7-amino-4-methylcoumarin-3-acetic acid, and beta-phycoerythrin. This method enables up to four separate antigens to be visualized within a single cell and two additional antigens to be detected in unrelated cells. The technique is illustrated by examining the cellular patterns of expression of liver fatty acid binding protein/human growth hormone fusion genes in the intestinal epithelium of adult transgenic mice. It should be generally applicable to other experimental systems that require localization of multiple antigens in single tissue sections.
Animals↗
A novel approach to the quantification of hepatic stellate cells in intravital fluorescence microscopy of the liver using a computerized image analysis system.
Hepatic stellate cells (HSC) are nonparenchymal liver cells which reside in the space of Disse within the hepatic microcirculatory unit. HSC can be distinguished using intravital fluorescent microscopy (IVFM) due to the autofluorescence from their intracellular vitamin A. Herein we report on a novel approach for the quantification of video-recorded rat HSC images acquired by IVFM (excitation 360 nm/emission 420 nm) by the combined use of the "Cell counting macro" and the "Measurement macros" in the NIH image software. The approach involved two major steps using (i) the "Cell counting macro" for automatic detection, threshold-setting, and generation of a binary image of the vitamin A autofluorescence in the HSC images and (ii) the "Compute percent black and white" command in the "Measurement macros" to automatically determine the HSC density (%), which was then expressed as percentage of the total area of vitamin A autofluorescence-associated sites per observation area. Comparing the vitamin A autofluorescence areas in the original and the binary fashion HSC images revealed that the "Cell counting macro" was an optimal option for the analysis of the low-magnification (x10 objective) HSC images, whereas this macro was not suitable for the analysis of the higher magnification (40x objective) HSC images unless modifications were made. Our analysis revealed that HSC represent approximately 4-5% of the total area of the liver surface. In analyzing the higher magnification HSC microfluorographs, the use of the original "Cell counting macro" resulted in a significant underestimation of HSC density (60% reduction, P < 0.01) when compared with those analyzed using our modified macro. This study represents the first report of an automatic and reliable approach to the intravital fluorescent microscopic quantification of HSC using a computer-NIH image analysis system.
Animals↗
Measurement of cochlear blood flow: intravital fluorescence microscopy.
A technique is described for directly observing in vivo cochlear microvasculature in the gerbil for physiologic and experimentally induced changes in vessel diameter and blood flow velocity. Measurements are made from computer processed video images of surgically exposed microvessels. These images are obtained using intravital fluorescence microscopy (IFM) with epi-illumination. The Mongolian gerbil is an ideal animal model for circulatory studies of the inner ear. It has a stable heart rate and blood pressure under urethane/alpha-chloralose anesthesia and its cochlea is surgically accessible. A window is created over the feeding artery (anterior inferior cerebellar artery) and over the stria vascularis of the second turn of the cochlea, atraumatically exposing radiating arterioles and strial capillaries. Our system of IFM provides images that are videorecorded, digitally analyzed with a computer image processor, and enhanced according to the type of measurement desired. Velocity measurements are obtained by tracking plasma gaps or single fluorescent labeled red blood cells through successive frames of the videorecorded images. This experimental technique allows us to analyze circulatory responsiveness to a variety of vasoactive drugs administered regionally to the cochlea in concentrations not affecting systemic circulation.
Animals↗
Localization of tetra(m-hydroxyphenyl)chlorin (Foscan) in human healthy tissues and squamous cell carcinomas of the upper aero-digestive tract, the esophagus and the bronchi: a fluorescence microscopy study.
To date, little is known about precise time-dependent distribution and histological localization of tetra(m-hydroxyphenyl)chlorin (mTHPC) in human healthy tissues and squamous cell malignancies in the upper aero-digestive tract. A fluorescence microscopy study was performed on 50 healthy tissue biopsies and on 13 tumors (graded from Tis to T1 SCC) from 30 patients. Tissue samples were taken between 4 h and 11 days following injection of 0.15 mg/kg mTHPC. A fairly comparable distribution pattern in various tissues was observed over time in different patients. Vascular localization of mTHPC fluorescence predominates at a short delay, whereas the dye is essentially located in the tumoral and healthy mucosa after longer delays. A much lower uptake and retention of mTHPC fluorescence was noted in striated muscle and cartilage as compared to neoplastic lesions. No significant selectivity was found between healthy and tumoral mucosa. The obtained data are important to confirm drug-light interval that have been selected for effective PDT for early SCC malignancies while minimizing the risks of over- or under-treatment. The low fluorescence level in striated muscle provides the opportunity to develop interstitial PDT as a treatment modality for invasive SCC of unfavorable locations in the oral cavity or pharynx, such as the base of the tongue.
Aged↗
Immunohistochemical localization and quantitative analysis of cellular glutathione peroxidase in foetal and neonatal rat tissues: fluorescence microscopy image analysis.
To quantitate the developmental changes in selenium-dependent cellular glutathione peroxidase during the perinatal period, tissue sections from foetal (day 12 to day 22) and neonatal (day 6) rats were stained immunohistochemically using specific polyclonal antiserum. The intensity of the staining was quantified by fluorescence microscopy image analysis. There was a general trend of enriched glutathione peroxidase in the epithelial linings and metabolically active sites. Significant fluorescence was detected in cardiomyocytes, hepatocytes, renal tubular epithelium, bronchiolar epithelium and intestinal epithelium at day 15. The intensity increased in a stepwise manner thereafter. The overall increase in the intensity of staining in the heart, liver, kidneys, lungs and intestine was 1.5-, 2.3-, 1.6-, 1.7- and 3.0-fold, respectively. The phase of most rapid increase occurred during the foetal period in the liver, intestine and heart. In the kidneys and lungs, glutathione peroxidase increased significantly during foetal life, and to a similar extent postnatally. These results suggest that the intracellular H2O2-scavenging system develops during the foetal period as an essential mechanism for living under atmospheric oxygen conditions. The late development observed in the kidneys and lungs is consistent with the relative biological immaturity of these organs in full-term neonates.
Animals↗
A technique for fluorescence microscopy in semithin sections.
We describe here a procedure to improve contrast and resolution in fluorescence microscopy of sectioned tissues. Tissue fragments were fixed in ethanol-glacial acetic acid, embedded in diethylene glycol distearate, and semithin sectioned. This method maintains tissue antigenicity while preserving the structure of cells and tissues. The thinness of the sections eliminates scattered and emitted light from tissue structures outside the plane of focus. The procedure is simple and quick, and works excellently with fluorescein-conjugated lectins and antibodies.
Animals↗
[EARLY DIAGNOSIS OF CARCINOMA OF THE UTERUS BY MEANS OF FLUORESCENT MICROSCOPY].
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Cytodiagnosis↗
Assessment of telomere length in hematopoietic interphase cells using in situ hybridization and digital fluorescence microscopy.
Telomeres are G/C-rich repetitive DNA sequences at the end of all eukaryotic chromosomes. The loss of telomeric repeat sequences during cell divisions has been proposed as a possible mechanism for cell senescence. The standard procedure for measurement of telomere length is Southern blot (SB) hybridization with a telomere-specific probe. However, in using this technique no information can be obtained on variation in telomeric fragments due to interchromosomal, intrachromosomal, and intercellular differences. Lansdorp et al. (Hum Mol Genet 5:685-691, 1996) developed a method to measure individual telomeres, using in situ hybridization on metaphase chromosomes, employing peptide nucleic acid (PNA) probes and digital fluorescence microscopy. In this paper we describe a method that can be used to assess telomeric length in interphase cells. An algorithm was developed to measure the total intranuclear fluorescence in situ hybridization (FISH) signal, which features accurate correction for the local autofluorescence. Application of this methodology to samples of fetal liver, umbilical cord blood, and adult bone marrow cells showed a gradual decrease of average telomeric length. Southern blot analysis and PNA FISH measurements on chromosomes in the same samples showed similar results. Advantages of interphase measurements include the possibility of studying nonproliferating cells, thus avoiding selection and cell culturing.
Adult↗
Pressure tuning of the morphology of heterogeneous lipid vesicles: a two-photon-excitation fluorescence microscopy study.
We used a technique that allows us to visualize local and morphological changes of the membrane of more component giant unilamellar vesicles due to high pressure perturbation. Under these conditions, thermally induced processes are largely suppressed, and the bending rigidity and line tension are influenced by pressure-induced changes in lipid molecular packing and shape only. We studied the effect of pressure on the lateral organization and morphology of the model raft system DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)/sphingomyelin/cholesterol as well as of the fluid mixture POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)/DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine) by two-photon excitation fluorescence microscopy. The pressure-dependent experiments were carried out using a sample cell made from a thin fused silica capillary. The use of Laurdan as fluorescence label allowed us to also follow the lipid phase state by calculating the generalized polarization (GP) values of the vesicles and extracting their average value. During the compression cycle, a reduction in the volume of the vesicles is observed, accompanied by an increase of the average GP value, indicating an increasingly tighter packing of the lipids. Interestingly, the two systems studied show phenomena of budding and fission, and these at surprisingly low pressures of 200-300 bar. Moreover, these budding processes are not directly related to phase transitions to an overall ordered conformational state of the lipid membrane, which occur at much higher pressures. The topological changes of the lipid vesicles are irreversible and exhibit a different behavior depending on whether the pressure is increased or decreased. The results are discussed in light of the various contributions to the free energy functional of lipid vesicles. Finally, the biological relevance of these studies is highlighted.
Fluorescent Dyes↗