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At least 847 records · Page 47Linked to original sources
PubMed2002

Prolonged increase in ciliary beat frequency after short-term purinergic stimulation in human airway epithelial cells.

Stimulation of ovine airway epithelial cells with 10 microM ATP for 1 min at 25 degrees C transiently increased both cytoplasmic calcium (fura-2 epifluorescence microscopy) and ciliary beat frequency (CBF; differential interference contrast microscopy) with a similar time course. Identical purinergic stimulation of human airway epithelial cells at 25 or 35 degrees C, however, lead to an increase in CBF that outlasted the calcium transient at least 20 min. While a nitric oxide synthase inhibitor had no effect, pre-treatment of human cells with inhibitors of cAMP-dependent kinase (PKA), 10 microM myristoylated PKA-inhibitory peptide and 1 microM KT-5720, as well as an inhibitor of adenylyl cyclase, 1 mM SQ22536, blocked the prolonged, but not calcium-coupled CBF increase. Addition of PKA inhibitors after purinergic stimulation only partially reduced CBF from its elevated plateau. Prolonged CBF increases did not depend on adenosine production as 10 microM UTP had an effect similar to ATP and 8-sulphophenyl-theophylline did not block them. After increasing human CBF in a PKA-dependent manner to a stable plateau with forskolin (10 microM), ATP caused only a transient, calcium-coupled CBF increase. Calcium transients were necessary for both short-term and prolonged CBF changes as ATP failed to produce CBF increases after emptying calcium stores with 1 microM thapsigargin. These data suggest that in human, but not ovine airway epithelial cells, ATP-induced calcium transients activate a signalling cascade including adenylyl cyclase and PKA. The resulting prolonged CBF stimulation does not rely only on PKA activity, suggesting that the decay of CBF is influenced by ciliary phosphatase activity.

Adenine↗
PubMed1984

Light microscopic observations on the release of vesicles by isolated chromaffin cells.

Cultured bovine adrenal medullary chromaffin cells were stimulated with the secretogogues Ba2+ or carbamyl choline plus Ca2+. With video-enhanced contrast, differential interference contrast microscopy, small vesicles were found to appear on the cell surface during stimulation. The structures were of lower refractive index than the cytoplasm, and their appearance required several tenths of a second. The vesicles are thought to correspond to omega figures seen with electron microscopy due to exocytosis. Many of the structures disappeared within a few seconds, but some appeared to coalesce into larger structures. The large structures may lead to the vacuoles that have been demonstrated to be present following stimulation. The nature of the cellular elements responsible for the vesicle which appeared on the surface was not found with either differential interference or interference reflection microscopy. The simplest explanation is that the refractive index of the elements is similar to that of the cell, and therefore the elements cannot be seen.

Adrenal Medulla↗
PubMed1981

Cell-to-substrate contacts in an adhesion-defective mutant of Balb/c3T3 cells.

The cell-to-substrate contacts of the adhesion-defective mutant, AD6, have been examined by interference reflexion microscopy and compared with those of the wild-type Balb/c3T3 cell. The 2 cell-types differed in their ability to produce focal contacts with the substrate (10-15 nm separation distance). Only 10% of AD6 versus 92% of Balb/c3T3 cells formed these contacts. When present in AD6 the focal contacts were smaller and fewer in number per cell than in Balb/c3T3. Close contact with the substrate (approx. 30 nm separation distance) was formed by both cell types. The absence of the stronger focal contacts accounts for the reduced substrate adhesion of the mutant cells and for other phenotypic characteristics such as reduced spreading, rounded shape, altered pattern of movement, and absence of stress fibres. The reduced adhesion of the mutant has been attributed to a change in the pattern of glycoproteins exposed at the cell surface, which results from a defect in glycosylation. This suggests that normal glycosylation and correct exposure of one or more cell surface components are required for the formation of a specific substrate adhesion, the focal contact, and offers the possibility of identifying individual surface components involved in formation of this adhesion. Attention is drawn to zero-order minima originating from cell thickness and not cell-to-substrate separation distance in the interference reflexion image.

Animals↗
PubMedDate not supplied

Three-dimensional reconstruction and motion analysis of living, crawling cells.

Cell behavior is three-dimensional (3-D), even when it takes place on a flat surface. Migrating cells form pseudopods on and off the substratum, and the cell body undergoes height changes associated with a 1 min behavior cycle. Inside the cell, the nucleus has a 3-D migratory cycle, and vesicles move up and down in the z-axis as a cell locomotes. For these reasons, the two-dimensional (2-D) analysis of cellular and subcellular behavior is, in many cases, inadequate. We have, therefore, developed 3-D motion analysis systems that reconstruct the cell surface, nucleus, pseudopods, and vesicles of living, crawling cells in 3-D at time intervals as short as 1 s, and compute more than 100 parameters of motility and dynamics morphology at 1-s intervals. We are now in the process of developing a multimode reconstruction system that will allow us to reconstruct and analyze fluorescently tagged molecular complexes within the differential interference contrast-imaged subcellular architecture of a crawling cell. These evolving technologies should find wide application for a host of biomedical problems.

Animals↗
PubMed2000

Isolation of Cyclospora oocysts from fruits and vegetables using lectin-coated paramagnetic beads.

Published techniques for recovering parasites from fruit and vegetables are generally inadequate, with low and variable recovery efficiencies. Herein, we describe an improved method for analyzing fruit and vegetables for Cyclospora oocysts. The technique includes washing procedures, sonication, and separation using lectin-coated paramagnetic beads. Identification is by microscopy (differential interference contrast and fluorescence). Oocyst recovery efficiencies from mushrooms, lettuce, and raspberries were approximately 12%. Recovery efficiencies from bean sprouts were approximately 4%. Although no significant difference in recovery efficiency could be detected between samples processed using the lectin-coated beads and samples processed without this procedure, distinct advantages were apparent when the lectin-coated beads were used. A considerably smaller, cleaner final volume remained for microscopy, which increases the sensitivity of the technique and reduces operator time.

Cyclospora↗
PubMed2002

[Mode of epithelium-fibroblast interaction in mixed heterotypic cell cultures].

The interaction between epithelium (dog kidney epithelium MDCJ/clone 20) and fibroblasts (diploid human fibroblasts M19 and AG-1523) was studied in mixed heterotypic cell cultures. The mode of cell interaction depends on the manner of their collision. At collision of the epithelium lamella and the lateral side of fibroblast, the lamella was seen to creep under the lateral side to force back the fibroblast. At the frontal collision of epithelium and fibroblast lamellae, the mode of interaction depends on the local situation. With the presence of a free substratum around, the fibroblast formed a new lamella and moved aside from the place of collision. In the case, when the neighboring cells prevented fibroblast from moving, it migrated under the epithelium. In this work, we have first demonstrated the formation of specialized intercellular adhesions between epithelium and fibroblasts. The cultures were studied by phase contrast, interference reflection or video tape recording, using an image processing system (Hamamatsu). For studying adhesion, immuno-fluorescent methods were performed.

Animals↗
PubMed1999

Attachment of fibroblasts on smooth and microgrooved polystyrene.

In this study rat dermal fibroblasts (RDFs) were cultured on smooth or microgrooved (1-20 microm wide, 0.5-5.4 microm deep) substrates. Polystyrene microgrooved substrates were produced by solvent casting on molds that had been produced by photolithographic techniques. We investigated the attachment of RDFs with various analytical techniques. Light microscopy and image analysis showed that RDFs were oriented on most microgrooves. The rate of orientation effectively was increased by an increase of groove depth. An analysis of confluent layers of RDF showed that at confluency microgrooves were able to support greater numbers of cells. However, the largest numbers of cells were not found on the narrowest and deepest microgrooves even though such microgrooves have the largest total surface and induce the strongest alignment. Interference reflection microscopy (IRM) showed that the RDFs form focal adhesions where the cell membrane is only 10 nm from the substrate. IRM also showed that RDFs follow the contours of shallow and wide microgrooves but bridge the grooves on deeper and narrower ones. This could explain why such grooves are not able to increase the numerical cell adhesion to a greater degree. The absence of contact between cells and the bottom of the grooves is a very important factor in establishing contact guidance.

Algorithms↗
PubMed1984

Dipyridamole reduces the motility of 10T1/2 cells and disturbs their stress fibre pattern.

Dipyridamole (DPD), a widely used anti-thrombotic agent, inhibits in vitro directional migration of 10T1/2 mouse embryo cells in a dose dependent way. The inhibition is detectable at DPD concentrations comparable to those in human plasma after oral intake of the drug. Using a model for quantitative shape change analysis, it is demonstrated that DPD inhibits pseudopodal activity and stabilizes the outlines of 10T1/2 cells. The inhibition of pseudopodal activity is thought to be involved in the reduced migratory capacity and the decreased area occupied by DPD treated cells. Coomassie blue staining of detergent-extracted cells shows that stress fibre patterns in 10T1/2 cells are altered by DPD. In particular, highly regular, polygonal networks, reminiscent of those described in spreading cells, appear after DPD treatment. It is suggested that interference of DPD with the normal organization of the actin microfilament system accounts for its effects on spreading, motility, and growth of cultured cells, and possibly for part of its in vivo anti-thrombotic activity as well.

Animals↗
PubMed2005

High-energy phase-contrast X-ray imaging using a two-crystal X-ray interferometer.

To broaden the scope of phase-contrast X-ray imaging in biomedical applications, the energy of X-rays in a large-area imaging system using a two-crystal X-ray interferometer has been increased from 17.7 keV to 35 keV. The use of higher-energy X-rays makes it possible to observe larger samples having greater variation in density within shorter measurement periods, at higher spatial resolution, and at lower X-ray doses. After optimizing the imaging system for 35 keV X-rays, a 25 mm x 30 mm interference pattern with 50% visibility was generated at the Photon Factory. The system's capabilities are demonstrated by this pattern and a high-quality three-dimensional image of a rat heart.

Animals↗
PubMed1986

Estrogen- and tamoxifen-induced rearrangement of cytoskeletal and adhesion structures in breast cancer MCF-7 cells.

The cytoskeleton, the shape, and the adhesion complexes of MCF-7 breast carcinoma cells have been studied by fluorescence, phase contrast, and interference reflection microscopy. Cells have been grown in media containing different concentrations of estrogen and with or without the addition of the antiestrogen tamoxifen. The pattern of actin microfilaments and keratin intermediate filaments (tonofilaments) and the distribution of adhesion areas change as a function of the estrogen concentration. When cells are cultured in estrogen-deprived medium, they appear roundish and flattened and adhere firmly to the substratum, with multiple vinculin-positive adhesion plaques at their ventral surface. Upon stimulation with estrogen, these cells display pseudopodial cytoplasmic protrusions and ruffling membranes; in interference reflection microscopy the adhesion areas are mostly localized in these projections. A rearrangement of microfilaments and of tonofilaments in the cell projections and the formation of a dense network of keratin fibers takes place. Tamoxifen affects cellular shape and cytoskeletal arrangement in a way similar to that induced by estrogen. An effect of estrogen-receptor stimulation on the adhesion structures and on the rearrangement of intermediate and actin filaments (and accordingly of the shape and internal structure of breast cancer cells) can be suggested. Such an effect might be direct or mediated through unknown mechanisms; it seems, however, to be independent of the well known estrogenic effect on cell proliferation.

Actins↗
PubMed2003

Culturing of avian embryos for time-lapse imaging.

Monitoring morphogenetic processes, at high resolution over time, has been a long-standing goal of many developmental cell biologists. It is critical to image cells in their natural environment whenever possible; however, imaging many warm-blooded vertebrates, especially mammals, is problematic. At early stages of development, birds are ideal for imaging, since the avian body plan is very similar to that of mammals. We have devised a culturing technique that allows for the acquisition of high-resolution differential interference contrast and epifluorescence images of developing avian embryos in a 4-D (3-D + time) system. The resulting information, from intact embryos, is derived from an area encompassing several millimeters, at micrometer resolution for up to 30 h.

Animals↗
PubMed1999

Reorganization of filamentous actin and myosin-II in zebrafish eggs correlates temporally and spatially with cortical granule exocytosis.

The zebrafish egg provides a useful experimental system to study events of fertilization, including exocytosis. We show by differential interference contrast videomicroscopy that cortical granules are: (1) released nonsynchronously over the egg surface and (2) mobilized to the plasma membrane in two phases, depending upon vesicle size and location. Turbidometric assay measurements of the timing and extent of exocytosis revealed a steady release of small granules during the first 30 seconds of egg activation. This was followed by an explosive discharge of large granules, beginning at 30 seconds and continuing for 1-2 minutes. Stages of single granule exocytosis and subsequent remodeling of the egg surface were imaged by either real-time or time-lapse videomicroscopy as well as scanning electron microscopy. Cortical granule translocation and fusion with the plasma membrane were followed by the concurrent expansion of a fusion pore and release of granule contents. A dramatic rearrangement of the egg surface followed exocytosis. Cortical crypts (sites of evacuated granules) displayed a purse-string-like contraction, resulting in their gradual flattening and disappearance from the egg surface. We tested the hypothesis that subplasmalemmal filamentous (F-) actin acts as a physical barrier to secretion and is locally disassembled prior to granule release. Experimental results showed a reduction of rhodamine-phalloidin and antimyosin staining at putative sites of secretion, acceleration of the timing and extent of granule release in eggs pretreated with cytochalasin D, and dose-dependent inhibition of exocytosis in permeabilized eggs preincubated with phalloidin. An increase in assembled actin was detected by fluorometric assay during the period of exocytosis. Localization studies showed that F-actin and myosin-II codistributed with an inward-moving, membrane-delimited zone of cytoplasm that circumscribed cortical crypts during their transformation. Furthermore, cortical crypts displayed a distinct delay in transformation when incubated continuously with cytochalasin D following egg activation. We propose that closure of cortical crypts is driven by a contractile ring whose forces depend upon dynamic actin filaments and perhaps actomyosin interactions.

Actin Cytoskeleton↗
PubMed2004

Transversal phase resolved polarization sensitive optical coherence tomography.

We present a novel optical coherence tomography (OCT) method to measure backscattered intensity and birefringence properties (retardation and fast axis orientation) and apply it to imaging of human ocular tissue. The method is based on a Mach Zehnder interferometer, on transversal scanning, and on a polarization sensitive two-channel detection. A highly stable carrier frequency is generated by acousto-optic modulators (AOMs). This allows a phase sensitive demodulation by the lock-in technique. Since the recording of individual interference fringes is avoided by this method the amount of data to be recorded and processed is considerably reduced. We demonstrate this method on human cornea and anterior chamber angle and present, to the best of our knowledge, the first OCT images of retardation and fast axis orientation of the anterior chamber angle region in vivo.

Anterior Chamber↗
PubMed1977

Combined Nomarski interference contrast and immunofluorescent study of neuropathological specimens: CSF sediments and paraffin embedded brain tissues.

The direct immunofluorescent technique may be easily improved by the use of the Nomarski optics. This contrast allows accurate identification of fluorescent CSF cells and structures in formalin fixed paraffin embedded brain tissues; in the latter, the combined optical procedure is fruitfull in order to avoid fluorescent artifacts misinterpretation. Furthermore, it is emphazised that the conditions in which routine neuropathological specimens are removed and stored usualy does permit the application of the immunofluorescent technique.

Brain↗