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At least 757 records · Page 42Linked to original sources
PubMed2002

Orthogonal polarization spectral imaging versus intravital fluorescent microscopy for microvascular studies in wounds.

The aim of this study was to validate orthogonal polarization spectral (OPS) imaging against intravital fluorescence microscopy (IFM) for microvascular measurements in normal skin and during wound healing. Experiments were performed on the ears of hairless mice (N = 8). The diameter of arterioles and venules, red blood cell velocity in venules, and the functional capillary density were assessed under normal conditions using OPS imaging and IFM. After creation of a circular wound, these observations were repeated at the identical microvascular regions on days 4, 7, 10, and 15. Images were videotaped, and CapImage was used for off-line computer-assisted analysis. Using OPS imaging, the microcirculation of wounded skin in hairless mice could be observed successfully. The regression analyses against standard IFM revealed a significant (p < 0.001) correlation for measurements of all microcirculatory parameters investigated (venular diameter: r(2) = 0.98, N = 345; red blood cell velocity: r(2) = 0.51, N = 326; functional capillary density: r(2) = 0.44, N = 156). However, for diameter as well as for functional capillary density measurements, OPS imaging yielded lower absolute values compared with IFM. The authors were able to validate OPS imaging against IFM for the measurement of microvascular parameters in an animal model of skin wound healing. Such a device should now help to study the role of microcirculation in physiology and pathophysiology during wound healing in patients. First clinical investigations are promising.

Animals↗
PubMed1996

Bifunctional protein cross-linking reagents improve labeling of cytoskeletal proteins for qualitative and quantitative fluorescence microscopy.

Because permeabilization of the cell membrane is necessary to label intracellular proteins with most fluorescent probes, it is important to optimize the preservation and labeling of the proteins under study. We used qualitative and quantitative fluorescence microscopy to evaluate the effects of six different bifunctional protein cross-linking reagents and several extraction conditions on the labeling of filamentous actin with phalloidin and the immunolabeling of tubulin and gelsolin. The labeling of cytoskeletal and associated proteins can be significantly enhanced by the appropriate combination of bifunctional protein cross-linking reagents and extraction conditions. However, the conditions that give the most intense labeling vary depending on the label used. The greatest intensity of labeling with either phalloidin or antibodies was obtained with the intermediate-length cross-linker DSP. The two-step procedure of cross-linking with DSP and extracting in Triton X-100 in microtubule-stabilizing buffer containing DSP gives maximal labeling with phalloidin. Maximal labeling of gelsolin and tubulin with antibodies is obtained by extracting DSP-cross-linked cells with Triton in Hank's saline containing DSP. Therefore, DSP reproducibly improves preservation of both soluble and filamentous proteins for quantitative and qualitative studies by fluorescence microscopy.

Actins↗
PubMed2000

Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells.

This paper describes the combined use of atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRFM) to examine the transmission of force from the apical cell membrane to the basal cell membrane. A Bioscope AFM was mounted on an inverted microscope, the stage of which was configured for TIRFM imaging of fluorescently labeled human umbilical vein endothelial cells (HUVECs). Variable-angle TIRFM experiments were conducted to calibrate the coupling angle with the depth of penetration of the evanescent wave. A measure of cellular mechanical properties was obtained by collecting a set of force curves over the entire apical cell surface. A linear regression fit of the force-indentation curves to an elastic model yields an elastic modulus of 7.22 +/- 0. 46 kPa over the nucleus, 2.97 +/- 0.79 kPa over the cell body in proximity to the nucleus, and 1.27 +/- 0.36 kPa on the cell body near the edge. Stress transmission was investigated by imaging the response of the basal surface to localized force application over the apical surface. The focal contacts changed in position and contact area when forces of 0.3-0.5 nN were applied. There was a significant increase in focal contact area when the force was removed (p < 0.01) from the nucleus as compared to the contact area before force application. There was no significant change in focal contact coverage area before and after force application over the edge. The results suggest that cells transfer localized stress from the apical to the basal surface globally, resulting in rearrangement of contacts on the basal surface.

Biophysical Phenomena↗
PubMed2002

Activation of dynamin II by POPC in giant unilamellar vesicles: a two-photon fluorescence microscopy study.

The interaction of dynamin II with giant unilamellar vesicles was studied using two-photon fluorescence microscopy. Dynamin II, labeled with fluorescein, was injected into a microscope chamber containing giant unilamellar vesicles, which were composed of either pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or a mixture of POPC and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Binding of the fluorescent dynamin II to giant unilamellar vesicles, in the presence and absence of PI(4,5)P2, was directly observed using two-photon fluorescence microscopy. This binding was also visualized using the fluorescent N-methylanthraniloyl guanosine 5'-[gamma-thio]triphosphate analogue. The membrane probe 6-dodecanoyl-2-dimethylamine-naphthalene was used to monitor the physical state of the lipid in the giant unilamellar vesicles in the absence and presence of dynamin. A surprising finding was the fact that dynamin II bound to vesicles in the absence of PI(4,5)P2. Activation of the GTPase activity of dynamin II by pure POPC was then shown.

Dynamin II↗
PubMed1993

Co-localization of mu and delta opioid receptors on SK-N-SH cells detected by fluorescence microscopy using labeled anti-idiotypic antibodies.

Selective fluorescence labeling of opioid receptor subclasses on SK-N-SH cultured cells has been accomplished using labeled polyclonal anti-idiotypic antibodies along with subclass-selective opioid agonists (DPDPE, delta-selective; DAMGO, mu-selective) as blocking reagents. Labeling of the cells was examined using conventional fluorescence microscopy. Co-localization of mu- and delta-opioid receptors on SK-N-SH cells has been studied by double labeling fluorescence experiments. In agreement with our own, and other workers', previous observations on NG108-15 cells, a subpopulation of viable cells in asynchronous cultures are labeled. Among those SK-N-SH cells that are labeled, both subclasses of receptors are seen. On the basis of sequential blocking experiments we interpret our combined results to be consistent with a model where mu- and delta- binding sites reside on different subunits of a multimeric complex.

Antibodies, Anti-Idiotypic↗
PubMed1990

Analysis of cholesterol ester accumulation in macrophages by the use of digital imaging fluorescence microscopy.

Low density lipoprotein (LDL) induced accumulation of cholesterol esters was analyzed by the digital imaging fluorescence microscopy (DIFM) in murine tumor macrophages. To analyze cholesterol ester accumulation, P388D1 macrophages were incubated with increasing quantities of unmodified or acetylated human LDL, washed, and live stained with a lipophylic fluorescent dye Nile Red. The increase in fluorescence intensity was quantitatively determined by the interactive laser cytometer (ACAS 470) and compared with the accumulation of cellular cholesterol esters determined by the gas liquid chromatography. Correlation between the two methods was highly significant (r greater than 0.9, P less than 0.001). A good agreement between the two methods was also found in terms of sensitivity and reproducibility. With the use of 589 nm narrowband interference filter in the light path of emitted light the intensity of fluorescence correlated well with cellular cholesterol ester content even in the presence of relatively high concentrations of triglycerides. Therefore, digital imaging fluorescence microscopy appears to be a reliable method for quantification of cholesterol ester accumulation at the single cell level offering new possibilities of studying interactions between cells and cholesterol ester rich lipoproteins.

Animals↗