The cytoskeleton and substratum adhesion in chick embryonic corneal epithelial cells.
Explore the source record for details and available documents.
Animals↗
SEARCH · Search PubMed
Results for “Microscopy, Interference”
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.
Organization of extracellular matrix by chick embryonic corneal epithelial cells in culture and the role of fibronectin in adhesion.
Corneal epithelial cells from 15-day chick embryos produce an extensive extracellular matrix when cultured for up to 7 days on plastic, glass, and gelatin-coated substrata. Immunofluorescence studies revealed the presence of a fibrillar fibronectin matrix that became more extensive with time in culture. When cells were cultured in fibronectin-depleted medium little or no fibronectin was evident in the first 24 h, although the cells attached and spread normally when compared with controls. Considerably more fibronectin was associated with the cells after 48 h. Incubation of cells with [35S]methionine, followed by immunoprecipitation of cell extracts by specific anti-fibronectin antibody, confirmed that the cells synthesize fibronectin. Cells cultured in fibronectin-coated substrata were able to reorganize the fibronectin into fibrillar form as well as to endocytose fibronectin. It is suggested that cells 'comb' fibronectin from the substratum and organize it into fibrils. Interference reflection microscopy revealed no simple correlation between the distribution of fibronectin and focal contacts. However, some focal contacts seemed to lack fibronectin. In the presence of anti-fibronectin immunoglobulin G cells initially attached but failed to spread, and subsequently detached from glass and fibronectin-coated substrata. However, they attached and spread on collagen substrata. The results suggest that fibronectin is unnecessary for initial cell attachment but may be important in maintaining cell adhesion and spreading on certain substrata. However, it is not necessary for attachment and spreading on collagen. Cells on collagen may attach and spread directly on the substratum via specific collagen receptors or by using some other type of attachment factor(s). Electron microscopy revealed a densely staining fibrillar matrix between the basal cell layer and substratum as well as in the intercellular spaces between cells. Components of this matrix and the cell surface were strongly stained by ruthenium red, which indicates the presence of acidic groups such as glycosaminoglycans.
Animals↗
Monensin inhibits secretion of extracellular matrix and the spreading of mesoderm cells in the early chick embryo.
Chick embryos in culture were treated with the secretion-inhibiting ionophore monensin at the gastrulation stage of development. After treatment for 5 h at a concentration of 10(-5) M the embryos showed a drastic reduction in the tissue space between the upper and lower epithelia, and reduced spreading of the mesoderm cells that occupied this space. The basement membrane, to which many mesoderm cells are normally attached, showed varying degrees of disruption, which permitted blebbing of the overlying epithelium. Intracellularly, the treatment caused extensive vacuolization in all tissues. These results were consistent with the effects expected by removal of hyaluronic acid from the extracellular space, and this was confirmed by demonstrating a sharp reduction in glucosamine incorporation in this region. Examination of the effects of monensin on isolated mesoderm cells in culture using interference reflection microscopy indicated that the spreading of these cells was reduced independently of the changes in the extracellular matrix of the embryo. That this was probably due to the inhibition of cell surface fibronectin secretion was shown by demonstrating severe changes in the distribution of this glycoprotein using the immunofluorescent technique. It is concluded that the effects of monensin on this intact, developing system are due primarily to the disruption of hyaluronic acid secretion, but that disrupted fibronectin synthesis contributes to the reduced spreading of mesoderm cells.
Animals↗
Effect of phorbol myristate acetate and the chemotactic peptide fNLPNTL on shape and movement of human neutrophils.
The results show that the distinct types of shape produced by phorbol myristate acetate (PMA) and by chemotactic peptides (fNLPNTL) are associated with distinct types of neutrophil movement. Whereas the chemotactic peptide can induce front-tail polarity characterized by an expanding front, a contracted tail and preferential unidirectional movements of intracellular organelles, PMA can only elicit non-polar movements characterized by random formation and retraction of projections all over the surface, intracellular movements of organelles being ill-defined and changing in direction. Combined stimulation of human neutrophils with PMA and fNLPNTL results in a suppression of peptide-induced polarity and the formation of non-polar motile cells resembling those stimulated with PMA alone. The results suggest that the diacylglycerol-protein kinase C pathway may be instrumental in transducing or modulating signals to the locomotor apparatus of the cell. PMA-treated cells are, however, still capable of developing directional responses when appropriately stimulated. The findings lead to the hypothesis that distinct types of neutrophil movements are preferentially associated with distinct functions.
Chemotaxis, Leukocyte↗
The translocation of mitochondria along insect ovarian microtubules from isolated nutritive tubes: a simple reactivated model.
Nutritive tubes, the microtubule-based translocation channels that link the trophic tissue to the developing oocytes in the ovaries of hemipteran insects, have been isolated and examined using video-enhanced differential interference contrast microscopy. When viewed in this way the nutritive tubes are seen to fray into linear strands, which, on the addition of exogenous ATP, support the translocation of particles along their lengths. The movement is also seen with GTP but not AMP-PNP. It is not affected by the addition of inhibitors of dynein or of energy metabolism. Electron microscopy shows the strands to consist of bundles of parallel microtubules of different sizes and the moving particles to be mitochondria. Comparisons are drawn between the movement of mitochondria along isolated insect ovarian microtubules and the reported translocation of vesicles along microtubules from squid axoplasm. The simplicity of the insect system is emphasized. The fact that it can be isolated easily and characterized biochemically makes it potentially valuable for investigating microtubule-based translocation.
Animals↗
Cell adhesion to hydroxyl groups of a monolayer film.
We have studied cells on chemically defined monomolecular films of the long-chain alcohol docosanol. Langmuir-Blodgett films of the alcohol were deposited on glass coverslips, previously made hydrophobic with octadecyl groups. This gives films in which the alcohol headgroups face outwards to the water. Molecular orientation and film integrity were shown by a fluorescence adsorption test. Cell contacts on the films were observed in media without proteins by interference reflection microscopy (IRM) and the mechanics of detachment were examined by hydrodynamic shearing in a flow chamber. Cell contact with docosanol was compared with that on an adjacent area of octadecyl glass without a monolayer. Dictyostelium amoebae settled and spread on both docosanol and octadecyl glass, but little or no locomotion was seen on docosanol. On octadecyl glass the amoebae moved actively, forming ultrathin cytoplasmic lamellae, which look dark under IRM, and left distinctive trails of membranous debris. Hydrodynamic shearing showed that the amoebae stuck strongly to both surfaces and could not be removed from either at the maximum attainable wall shear stress of 6Nm-2. Red blood cells also adhered to both surfaces and removal from both occurred between 1 and 3Nm-2. IRM and scanning electron microscopy (SEM) studies indicated that this force leads to a minimal measure of red cell adhesion, since removal often involved the breakage of cytoplasmic tethers. Our results show that alcoholic -OH groups, in a two-dimensional array, provide a surface that is strongly adhesive for cells. No other method has made it possible to demonstrate cell adhesion purely to -OH groups, in a known orientation and density, and in the absence of any other functional groups on the interface.
Cell Adhesion↗
Microinterferometry of the movement of dry matter in fibroblasts.
We describe the use of interferometric microscopy coupled with a novel application of Sénarmont compensation for detecting and quantifying the distribution of dry matter in cultured cells. In conjunction with video techniques and digital image processing, a two-dimensional, calibrated map of the dry mass distribution in an isolated cell can be obtained and digitally recorded. We have called the technique Digitally Recorded Interferometric Microscopy with Analyser Shift (DRIMAS). The method greatly facilitates the automatic recognition of cells by computer. Recorded time-lapse sequences can be used to establish a database of the growth and motility of specific cells in given experimental conditions. Databases of this type can be analysed to reveal the patterns of growth and locomotory behaviour of individual cells. We describe a systematic method of obtaining parameters of cell size, shape, spreading, intracellular motility and translocation. Auto-correlations and cross-correlations between these parameters can be detected and quantified using time series analysis, revealing potential cause/effect relationships in the mechanisms of growth and motility. Besides characterizing the overall pattern of cell behaviour, these data can also yield information about the instantaneous pattern of intracellular motility. We describe the use of finite element analysis to reveal the dynamics of the intracellular transport of dry matter. This yields the pattern of the minimum flow of dry matter required to account for the changes in its distribution. Most of this flux is not associated with the movement of visible structures and possibly represents the transport of dissociated components of the cytoskeleton. In chick heart fibroblasts, surprisingly high velocities of nearly 2.0 microns s-1 were detected during the period of increased motility following tail detachment. The total kinetic energy associated with the dry mass flux is a single parameter, which characterizes the instantaneous motility of the cell. We found that the kinetic energy of intracellular motility can be several hundred times greater than the kinetic energy of translocation. Kinetic energy may prove to be a very informative single measure of intracellular motility for assessing the effects of malignant transformation, genetic manipulations, and other experimental treatments on the locomotory machinery of the cell.
Animals↗
Cold inhibition of cell volume regulation during the freezing of insect malpighian tubules.
Cells in freeze-tolerant tissues must survive substantial shrinkage during exposure to the hyperosmolarity that results as solutes are excluded from extracellular ice. We investigated the possibility that this hyperosmotic shock elicits an acute regulatory volume increase (RVI) by monitoring the response of epithelial cell volume in the Malpighian tubules of the New Zealand alpine weta (Hemideina maori) during exposure to low temperature/hyperosmolarity (mimicking freezing conditions) or during an actual freeze/thaw cycle. The cross-sectional area of cells in isolated Malpighian tubules was measured using differential interference contrast microscopy. At 20 degrees C, cells held in saline containing 400 mmol1-1 glucose exhibit an RVI in response to hyperosmotic shock. Cross-sectional area decreased by 30% immediately after a change from iso-osmotic (0.7 osmol1-1) to hyper-osmotic saline (2.1 osmol1-1, equal to the osmotic shock encountered during freezing to -4 degrees C) and then returned to 21% below the control value 30 min after the exposure. Although substantial cellular function of Malpighian tubules was retained at low temperature (the rate of fluid secretion by isolated tubules at 4 degrees C was 72% of that measured at 20 degrees C), no RVI was observed at 0% degrees C; cross-sectional area was 39% below the control value immediately after the hyperosmotic exposure and 36% below the control value 30 min after hyperosmotic exposure. Dibutyryl cyclic AMP potentiated the RVI observed at 20 degrees C, but failed to elicit an RVI at 0 degrees C. A substantial RVI was also absent when the saline contained trehalose rather than glucose, regardless of whether the tubules were held at 20 degrees C or 0 degrees C. The cross-sectional area of cells in saline containing glucose remained at approximately 30% below the control value during an entire 30 min period of actual freezing to -4 degrees C, suggesting that an acute volume regulatory response was in fact inhibited during mild freezing. The inhibition of an acute RVI during mild freezing may serve to avoid the energetic expenditure associated with volume regulation at a time when the normal defence of cell volume appears to be unnecessary.
Animals↗
Desiccation survival of the infective larva of Haemonchus contortus.
Explore the source record for details and available documents.
Animals↗
Water uptake and hatching in the potato cyst nematode, Heterodera rostochiensis, and the root knot nematode, Meloidogyne incognita.
Explore the source record for details and available documents.
Animals↗
[Flow-induced Ca2+ transients regulate morphological changes in vascular endothelial cells via calpain-mediated proteolytic cleavage].
Explore the source record for details and available documents.
Calcium↗
Effects of protein transport inhibitors on the distribution and secretion of the fusion protein RntA-EGFP in Aspergillus oryzae.
The distribution of the secreted protein ribonuclease T1 (RntA) fused with the enhanced green fluorescent protein (EGFP), RntA-EGFP, was visualized in hyphae of Aspergillus oryzae in the presence of a protein transport inhibitor, brefeldin A, cytochalasin A, or nocodazole. During treatment with the protein transport inhibitors, the distribution of RntA-EGFP changed and distinct patterns of fluorescence accumulation were observed. The addition of brefeldin A caused RntA-EGFP fluorescence to appear in reticular networks, and the disruption of the polymerization of actin filaments by cytochalasin A caused an increase in RntA-EGFP fluorescence intensity in the hyphae without accumulation in a specific cellular component. In contrast, RntA-EGFP fluorescence was distributed in different parts of a hypha during treatment with nocodazole, a compound that depolymerizes microtubules. In addition, quantitative analysis was performed using the RntA-EGFP visualization system to analyze the relative amount of RntA-EGFP secreted into the culture medium during treatment with the protein transport inhibitors.
Aspergillus oryzae↗
Common-path interferometer for frequency-domain optical coherence tomography.
A Michelson-type spectral interferometer that uses a common beam path for the reference and the sample arms is described. This optical arrangement is more compact and stable than the more commonly used dual-arm interferometer and is well suited for frequency-domain optical coherence tomography of biological samples. With a 16-bit CCD camera, the instrument has sufficient dynamic range and resolution for imaging to depths of 2 mm in scattering biological materials. Images obtained with this spectral interferometer are presented, including cross-sectional images in a Xenopus laevis tadpole.
Abdomen↗
Phase-slope and group-dispersion calculations in the frequency domain by simple optical low-coherence reflectometry.
We report a new method by which phase slope and group dispersion can be calculated with a simple optical low-coherence reflectometer to quantify physiological conditions. A discrete-time signal processing algorithm based on the first and second derivatives of the phase with respect to wave number was developed from discrete-time Fourier properties. The algorithm avoids the 2pi ambiguity associated with most phase unwrapping. Experimental data collected by use of well-characterized optical materials validated the algorithm, which was minimally sensitive to phase noise. The group dispersion of bovine cornea was measured at various hydrations and was significantly dependent on hydration. The results suggest that group dispersion is an indicator of corneal alterations.
Algorithms↗
Full-field optical coherence tomography by achromatic phase shifting with a rotating polarizer.
We demonstrate two-dimensional detection optical coherence tomography (OCT) using achromatic phase shifting with a rotating polarizer. This phase shifting, which experiences a light beam with a cyclic change in its polarization state, is, in principle, independent of wavelength. We simulated the wavelength dependence of an achromatic phase shifter using Jones calculus and found that the achromatic region exceeded 145 nm when the deviation of the phase retardation was less than +/- 0.5 degrees. Using the achromatic phase shifter and a conventional phase-shift calculation method, we obtained en face OCT images of an onion at different depths. This method is effective to enhance the quality of OCT with an ultrabroad-spectrum light source.
Equipment Design↗
Micromachined array tip for multifocus fiber-based optical coherence tomography.
High-resolution optical coherence tomography demands a large detector bandwidth and a high numerical aperture for real-time imaging, which is difficult to achieve over a large imaging depth. To resolve these conflicting requirements we propose a novel multifocus fiber-based optical coherence tomography system with a micromachined array tip. We demonstrate the fabrication of a prototype four-channel tip that maintains a 9-14-microm spot diameter with more than 500 microm of imaging depth. Images of a resolution target and a human tooth were obtained with this tip by use of a four-channel cascaded Michelson fiber-optic interferometer, scanned simultaneously at 8 kHz with geometric power distribution across the four channels.
Equipment Design↗
Noncontact measurement of nerve displacement during action potential with a dual-beam low-coherence interferometer.
We have used a novel phase-referenced heterodyne dual-beam low-coherence interferometer to perform what we believe are the first noncontact measurements of surface motion in a nerve bundle during the action potential. Nerve displacements of approximately 5-nm amplitude and approximately 10-ms duration are measured without signal averaging. This interferometer may find general application in measurement of small motion in cells and other weakly scattering samples.
Action Potentials↗
Simple approach to one-laser, broadband coherent anti-Stokes Raman scattering microscopy.
Coherent anti-Stokes Raman scattering (CARS) microscopy is emerging as a powerful method for imaging materials and biological systems, partly because of its noninvasiveness and selective chemical sensitivity. However, its full potential for species-selective imaging is limited by a restricted spectral bandwidth. Recent increases in bandwidth are promising but still are not sufficient for the level of robust component discrimination that would be needed in a chemically complex milieu found, for example, in intracellular and extracellular environments. We demonstrate a truly broadband CARS imaging instrument that we use to acquire hyperspectral images with vibrational spectra over a bandwidth of 2500 cm(-1) with a resolution of 13 cm(-1).
Equipment Design↗