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PubMed1986

Fluorescent microscopy of hematoporphyrin derivative in the nude mouse tumor model.

Mechanisms for localization of hematoporphyrin derivative (HPD) within malignant tissue and for tumor necrosis after photodynamic therapy have not yet been established with certainty. We describe a modified freeze-drying technique to study these mechanisms. Normal tissues and squamous cell carcinoma xenografts were examined in nude mice after administration of HPD. Skin reveals marked fluorescence in connective tissue cells. No fluorescence is visible in surface epithelial cells or keratin. Liver shows diffuse fluorescence only in cells lining sinusoids. In tumor, malignant cells do not fluoresce and connective tissue cells fluoresce brilliantly. This technique provides a clear view of HPD fluorescence, frozen instantaneously in location and time. Fluorescence from connective tissue cells in skin and tumor suggests that localization and photodynamic action may be targeted in part at cells that critically support malignant epithelial cell growth as well as at malignant epithelial cells directly.

Animals↗
PubMed2006

Quantitative fluorescence microscopy: from art to science.

A substantial number of elegant experimental approaches have been developed to image the distribution and dynamics of DNA, mRNA, proteins, organelles, metabolites, and ions in living plant cells. Although the human brain can rapidly assimilate visual information, particularly when presented as animations and movies, it is much more challenging to condense the phenomenal amount of data present in three-, four-, or even five-dimensional images into statistically useful measurements. This review explores a range of in vivo fluorescence imaging applications in plants, with particular emphasis on where quantitative techniques are beginning to emerge.

Gene Expression↗
PubMedDate not supplied

Magnetic resonance spectroscopy and fluorescence microscopy to investigate mobile lipids in sensitive, resistant and reverting K562 cells and their membranes.

The erythroleukaemic K562 cell line and its adriamycin resistant counterpart were used to study resistance, its reversion and their consequences on the levels and localisation of lipids detected in proton nuclear magnetic resonance (NMR) spectra. On whole cells, the mobile lipids giving rise to a NMR signal were significantly decreased in the resistant cells when compared to the sensitive ones; these lipids recovered partially in the reverting cells. According to the spinlattice relaxation times (T1), the lipids detected appeared to be in a similar environment in sensitive and reverting cells. In membrane-enriched fractions, mobile lipid levels were not significantly different in the sensitive and reverting cell lines but decreased in resistant ones. Moreover, lipid droplets stained with a fluorescent Nile red lipophilic probe showed the presence of highly fluorescent particles in the samples in which NMR detected high levels of mobile lipids. These results suggest the participation of cytosolic lipid droplets in NMR signals in drug sensitive and reverting cells and open the question of the relative roles of these droplets and of the membrane lipids in the lipid metabolic pathways associated with drug resistance in cancer cells.

Drug Resistance, Multiple↗
PubMed2004

Apertureless scanning near-field fluorescence microscopy in liquids.

We show that apertureless scanning near-field optical microscopes that use sharp vibrating conical tips can be operated in liquid environments. We have investigated the damping of the tip oscillation as a function of its shape and as a function of its depth under the liquid surface. The degradation of the quality factor from 150 in air down to 15 in liquid does not impede to perform topographic and optical measurements with a very good sensitivity. As an example of application, we present near-field fluorescence images of dye-doped polystyrene spheres immersed in a liquid.

Microscopy, Fluorescence↗
PubMed1984

Catecholamine-synthesizing enzymes in paraganglia of aged Fischer-344 rats. Immunohistochemistry and fluorescence microscopy.

The catecholamine-synthesizing enzymes, tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase were examined by immunohistochemistry in hypertrophied paraganglia of aged male Fischer-344 rats. All paraganglionic cells reacted with antibodies against tyrosine hydroxylase. Dopamine beta-hydroxylase was identified in most paraganglionic cells, indicating that they synthesized norepinephrine. A variable number of paraganglia were positive for phenylethanolamine-N-methyltransferase, which suggested that they synthesized epinephrine. The formaldehyde-induced fluorescence method demonstrated greenish-yellow fluorescence or yellowish-brown fluorescence. The intensity of the fluorescence was in the same range as in adrenal medullary cells. The observations indicate that paraganglia are capable of synthesizing epinephrine.

Animals↗
PubMed1999

Comparison of fluorescence microscopy and culture assays to quantitate adhesion of Porphyromonas gingivalis to mono- and multi-layered pocket epithelium cultures.

BACKGROUND: The present study compared 2 different methods (direct versus indirect evaluation) for the quantification of the adhesion of Porphyromonas gingivalis strains to in vitro cultured mono-layers of pocket epithelium. METHODS: The indirect culture viability assay (calculation of colony forming units) was compared to a direct microscopic evaluation using a novel fluorescent stain. The fluorescent kit was found to stain both bacteria and epithelial cells and enabled a differentiation between dead and living cells. RESULTS: Comparing the visual to the culture data, a high and significant correlation was found (Pearson's correlation = 0.75; P <0.001). The adhesion capacity was in general higher for dead epithelial cells than for living cells (P <0.01). Although comparable numbers of bacteria of 2 P. gingivalis strains (Pg 4 and Pg 5) were applied, Pg 4 showed a significantly lower adhesion capacity. This intra-strain variability was observed by the culture assay (2.3 x 10(6) versus 7.8 x 10(6)+/-2.7 x 10(6); P <0.01) and by the direct microscopy (P <0.01) for both live and dead epithelial cells. A second goal was to see whether there was a difference in the amount of bacterial adherence to mono- and multi-layers of in vitro cultured epithelium. No significant differences were found for the 5 examined P. gingivalis strains. However, interstrain differences in adhesion capacity were evident for both tissues. CONCLUSIONS: This study highlights the reproducibility of a direct microscopic evaluation of bacterial adhesion to in vitro cultured epithelial cells, and suggests both intrastrain (P. gingivalis) and inter-cell (live versus dead) variation in adhesion capacity. Studies are needed to determine the extent to which P. gingivalis strain variation is reflected in variation of other strains in humans.

Bacterial Adhesion↗
PubMed2000

Visualization of flow-dependent concentration polarization of macromolecules at the surface of a cultured endothelial cell monolayer by means of fluorescence microscopy.

To substantiate the occurrence of flow-dependent concentration or depletion of atherogenic lipoproteins, which has been theoretically predicted to take place at a blood/endothelium boundary, we have studied the effects of perfusion pressure and wall shear rate on the accumulation and uptake of microspheres by cultured vascular endothelial cells in a monolayer. The study was carried out by flowing a cell culture medium containing fetal calf serum and fluorescent microspheres through a parallel-plate flow chamber having a cultured bovine aortic endothelial cell (BAEC) monolayer on one wall of the chamber. The microspheres had a nominal diameter of 19 nm, approximately the same as that of low-density lipoproteins, and thus served as models and tracers of plasma proteins and lipoproteins. Experiments were carried out in steady flow in the physiological range of wall shear rate and water filtration velocity at the monolayer, while monitoring the intensity of fluorescence of the spheres accumulated at and taken up by the endothelial cells. It was found that in a perfusate containing only fluorescent microspheres, due to increased phagocytic activity of the endothelial cells, the intensity of fluorescence which reflected the number of the microspheres taken up by the endothelial cells, increased almost linearly with time and independently of wall shear rate. However, with perfusates containing fetal calf serum, this abnormal phenomenon did not occur, and the intensity of fluorescence increased with increasing perfusion pressure and decreasing wall shear rate. It was also found that the number of fluorescent microspheres accumulated at and taken up by the BAEC monolayer was shear-dependent only at low wall shear rates, and increased sharply when the flow rate was reduced to zero. These results provided solid experimental evidence that flow-dependent concentration or depletion of macromolecules occurs at the luminal surface of the endothelium at physiological wall shear rates and water filtration velocities, and strongly supports the hypothesis that flow-dependent concentration polarization of lipoproteins plays an important role in the localization of atherosclerosis and intimal hyperplasia in man by facilitating the uptake of atherogenic lipoproteins by endothelial cells.

Animals↗
PubMed1994

Binding of anti-trifluoroacetyl antibodies to isolated hepatocytes observed by digital fluorescence microscopy.

These experiments were designed to observe specific binding of fluorescein-conjugated FAB'2 secondary antibodies to epitopes on the surface of isolated hepatocytes. The hepatocytes were attached as monolayers on microscope cover slips and an antigenic adduct known to be formed during metabolism of halothane, -trifluoroacetyl-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, was exchanged into their surface. Then the monolayers of hepatocytes were incubated with primary rabbit antibodies specific for the trifluoroacetyl group. Each coverslip was mounted in a perfusion chamber on a fluorescence microscope and a set of digital fluorescence images was made. Then fluorescein-conjugated goat-anti-rabbit FAB'2 secondary antibodies were flowed over the monolayer, the perfusion chamber was washed with buffer, and a second set of digital fluorescence images was made. The difference of these two sets of images demonstrated intense fluorescence superimposed on the outline of the cells. This intense fluorescence was not observed in control experiments in which the primary antibodies were omitted.

Animals↗
PubMed2002

Fluorescence microscopy imaging of bone for automated histomorphometry.

We have developed a computer-based method for the automated quantification of bone tissue in histological sections of decalcified specimens. Bone tissue was generated by ectopic implantation of ceramic-based carriers loaded with human bone marrow stromal cells (BMSCs). The method is based on the acquisition of multimodal images, in order to identify and measure the area covered by bone tissue (using fluorescent light) and the total area of tissue (using transmitted light), thereby excluding the regions corresponding to nonresorbed scaffold. The amount of bone as a percentage of the total area of interest (bone/area) and of the newly formed tissue (bone/tissue) is automatically derived. The computer-based results correlated closely with those obtained by manual identification of bone and tissue areas in the same histological fields (R(2) = 0.997; p < 0.0005), with errors dependent on the magnification used but always lower than 9.4%. The method was used to compare the bone/tissue and bone/area percentages in samples of engineered bone based on human BMSCs expanded in the presence of different biochemical factors and loaded onto different scaffolds. The technique thus represents a valuable tool to quantify reproducibly, accurately, and easily bone formation in a variety of tissue-engineering studies.

Bone Marrow Cells↗
PubMed2006

Implementation of X-ray fluorescence microscopy for investigation of elemental abnormalities in amyotrophic lateral sclerosis.

The abnormalities of metallochemical reactions may contribute to the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). In the present work, an investigation of the elemental composition of the gray matter, nerve cells and white matter from spinal cord tissues representing three ALS cases and five non-ALS controls was performed. This was done with the use of the synchrotron microbeam X-ray fluorescence technique (micro-SRXRF). The following elements were detected in the tissue sections: P, S, Cl, K, Ca, Fe, Cu, Zn and Br. A higher accumulation of Cl, K, Ca, Zn and Br was observed in the nerve cell bodies than in the surrounding tissue. Contrary to all other elements, Zn accumulation was lower in the white matter areas than in the gray matter ones. The results of quantitative analysis showed that there were no general abnormalities in the elemental accumulation between the ALS and the control group. However, for individual ALS cases such abnormalities were observed for the nerve cells. We also demonstrated differences in the elemental accumulation between the analyzed ALS cases.

Amyotrophic Lateral Sclerosis↗
PubMed1973

Identification of malaria parasites by fluorescence microscopy and acridine orange staining.

The need for a technique that is more sensitive than the use of Romanowsky-stained thick blood films for detecting malaria parasites at low concentration in the blood is well recognized. One of the more promising methods appeared to be fluorochrome staining with acridine orange. However, reports on the efficacy of the technique were contradictory and it was not clear to what extent blood films taken under survey conditions would contain fluorescing artefacts that might confuse diagnosis. An investigation indicated that, provided reasonable care was taken, blood films made under survey conditions contained few confusing artefacts. However, it was found that, while acridine orange staining might have a slight advantage when large malaria parasites were present, it was inferior to routine Romanowsky staining for the detection of young trophozoites, the inferiority becoming more pronounced as the parasite concentration decreased.

Acridines↗
PubMed2002

Ultrasensitive pharmacological characterisation of the voltage-gated potassium channel K(V)1.3 studied by single-molecule fluorescence microscopy.

The determination of pharmacologically relevant constants is crucial in order to understand the effects of compounds interacting with various membrane receptors. In this report we study a venom component of the Central American scorpion Centruroides limbatus, a short peptide termed hongotoxin(1) (HgTX(1)), which specifically binds to the voltage-gated potassium channel K(V)1.3 at a molecular stoichiometry of 1:1. A toxin analogue (HgTX(1)-A19C) was subjected to fluorescence labelling studies with Cy5. Utilising an ultrasensitive microscopic method (single-dye tracing; SDT) we were able to directly visualise HgTX(1)-A19C-Cy5 binding to the voltage-gated potassium channel K(V)1.3 on Jurkat cells at the single molecule level. For the first time, this approach allowed the determination of both the dissociation constant (K(D)) and the off-rate (k(off)) of HgTX(1)-A19C-Cy5 on living cells. In order to validate this novel approach, the data obtained with SDT were correlated to radioligand binding studies performed under identical conditions using a radioiodinated HgTX(1) analogue.

Binding, Competitive↗
PubMed1982

Immobilized thylakoids in a cross-linked albumin matrix: effects of cations studied by electron microscopy, fluorescence emission, photoacoustic spectroscopy, and kinetic measurements.

Immobilization of lettuce (Lactuca sativa) thylakoids has been performed by using glutaraldehyde and bovine serum albumin. Confirming previous reports, a stabilization of the O(2) evolution activity of the photosystem II (PSII) under storage and functional conditions has been observed. The present work is devoted to the role played by mono-and divalent cations, during the immobilization process itself, on the O(2) production. Four types of measurements have been employed: kinetic measurements, low temperature (77 K) fluorescence emission, photoacoustic (PA) spectroscopy, and electron microscopy observations. We show that the effect of glutaraldehyde is complex because it acts as an inhibitor, a stabilizing agent, and a cross-linking reactive. In the present studies, the thylakoids are immobilized within a polymeric insoluble albumin matrix. The highest activity yield and the best storage conditions are obtained when 0.15 mm Na(+) (or K(+)), 1 mm Mg(2+), and 0.1 mm Mn(2+) are present in the resuspending media before the immobilization. Due to modifications of the ionic content during such a process, structural differences are observed on the stacking degree of thylakoids. No modification of the fluorescence and PA spectra after the immobilization are found. Furthermore, a correlation between activities and spectral changes have been shown: when the activities increase, the F(735) to F(695) ratio increases and the PA(676) to PA(440) ratio decreases.

Journal Article↗
PubMed1995

Increased capillary endothelial leakage in portal hypertensive gastric mucosa: fluorescence microscopy in CCl4-induced cirrhotic rats.

Since portal hypertension affects the gastric mucosa, leading to congestive gastropathy and thus to increased incidence of bleeding, it is one of the possible causes of increased permeability of gastric mucosal capillaries. The aim of this study was the quantitative assessment of the permeability of the gastric mucosal endothelial cells. Eight CCl4-induced cirrhotic rats and eight matched controls were subjected to i.v. injection of FITC-albumin, and a morphometric evaluation of fluorescence in serial histological sections of the gastric mucosa was performed by a video image analysis system. Fluorescence was found to be 0.351 +/- 0.01% of the area scanned in experimental animals versus 0.073 +/- 0.005% in controls, i.e. it was significantly increased by the treatment, which implies a significant endothelial leakage into the extravascular space.

Albumins↗
PubMed1985

Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy.

We studied the laminar organization of 3T3 fibroblast cells growing on glass slides by use of total internal reflection illumination to excite fluorescence emission (TIRF) from labeled molecules and stained cellular compartments that are very close to the cell-substrate contact region. Mitochondria, distant from the contact regions and stained with the water-soluble cationic dye, dil-C3-(3), fluoresced only as the glass/cytoplasm critical angle was approached. A similar result was obtained when the nuclei were stained with Hoechst dye 33342. From this measured angle a cytoplasmic refractive index in the range 1.358-1.374 was computed. The plasma membrane of 3T3 cells was stained with dil-C18-(3), and the cytoplasmic compartment was stained with fluoresceinyl-dextran (FTC-dextran) or with carboxyfluorescein. We have demonstrated a high degree of correspondence between the low-reflectance zones in the reflection interference image of a live cell and the TIRF images of both the plasma membrane and cytoplasmic compartment. TIRF photometry of selected contact regions of cells provided data from which the absolute separation of cell and substrate was computed. From a population of 3T3 cells microinjected with fluorescein-labeled actin, motile and adherent interphase cells were selected for study. For adherent cells, which displayed fluorescent stress fibers, the TIRF image was composed of intense patches and less intense regions that corresponded, respectively, to the focal contact and close-contact zones of the reflection-interference image. The intense patches corresponded to the endpoints of the stress fibers. Cells of motile morphology, which formed some focal contacts and extensive close-contact zones, gave AF-actin TIRF images of relatively even intensity. Thin lamellar regions of the cytoplasm were found to contain concentrations of actin not significantly different from other close-contact regions of the cell. The major analytical problem of TIRF microscopy is separation of the effects of proximity to substrate, refractive index, and fluorescent probe concentration on the local brightness of the TIRF image. From our results, it appears possible to use TIRF microscopy to measure the proximity of different components of substrate contact regions of cells.

Actins↗
PubMed1974

[Uptake of methyl-12-benzo (a) and methyl-7-benzo (c) acridines by target organs of the female rat studied by fluorescence microscopy (author's transl)].

The diffusion and uptake of two benzacridines with very similar chemical structures, but with different carcinogenic powers were investigated at the level of various organs and at the cellular level in the female rat. The chronology of the processes and lesions observed was different according to the respective carcinogenic power of the two benzacridines. On account of the small doses used, only necrotic lesions were observed, mainly in the liver and adrenal glands, but no tumorous lesions were observed. Uptake of fluorescent substance at the level of the nuclei of certain cells (submaxillaris, liver, pancreas, adrenal glands) was noticed as in previous experiments; this transient process appeared about the 100th day and lasted fairly long according to the form of benzacridine and the organ.

Acridines↗
PubMed2004

Automatic real-time three-dimensional cell tracking by fluorescence microscopy.

Live cell imaging has become an indispensable technique for cell biologists. However, when grown on coverslip glass used for live cell imaging many cultured cells move even during relatively short observation times and focus can drift as a result of mechanical instabilities and/or temperature fluctuations. Time-lapse imaging therefore requires constant adjustment of the imaging field and focus position to keep the cell of interest centred in the imaged volume. We show here that this limitation can be overcome by tracking cells in a fully automated way using the mass centre of cellular fluorescence. Combined with automated multiple location revisiting, this method dramatically increases the throughput of high-resolution live cell imaging experiments.

Algorithms↗
PubMed1981

Glucose oxidase immunoenzyme methodology as a substitute for fluorescence microscopy in the clinical laboratory.

Enzymes as markers for antigens or antibodies in immunohistochemical procedures have several advantages over commonly used fluorochrome labels. These include use of a regular light microscope and the ability to get permanently stable slide preparations. Glucose oxidase (EC 1.1.2.3.4), being absent in mammalian tissue, provides no background staining, such as that seen with the commonly used horseradish peroxidase (EC 1.11.1.7) owing to peroxidase-like activity in tissues. A glucose oxidase histochemical method is detailed that is useful for detection of human antibodies; it can be easily used in clinical laboratories as a substitute for fluorescent techniques.

Animals↗