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At least 199 records · Page 11Linked to original sources
PubMed2000

Phagocytosis of yeast: a method for concurrent quantification of binding and internalization using differential interference contrast microscopy.

In studies of phagocytosis there is a need to distinguish targets that are internalized by the cell from those that are bound to the cell surface. The present work describes a simple method by which internalized and surface-bound yeast particles can be identified by differential interference contrast microscopy, using trypan blue to stain surface-bound yeast particles. The method has the advantage that both internalized and surface-bound particles can be visualized without the need to switch the illumination source and/or filter sets, thus facilitating concurrent quantitation of binding and internalization. The method was evaluated with the phagocytosis-modulating agents horseradish peroxidase (HRP) and cytochalasin D, using adherent resident macrophages as phagocytic cells. When macrophages are challenged with a particular type of target, they usually bind many more targets than they ingest. It was shown that yeast particles were arrested in the initial binding phase of phagocytosis depending on the region of macrophage plasma membrane where binding sites were formed. Failure of surface-bound yeast particles to trigger internalization was not due to modifications of the yeast particle surface. Nor was it due to binding to non-phagocytic receptors, or low-affinity receptor-ligand interactions. The glycoprotein HRP inhibited only the binding stage of phagocytosis, whereas cytochalasin D, a drug that affects actin polymerization, inhibited both binding and internalization. However, when the yeast particles were pre-incubated in fresh mouse serum, cytochalasin D inhibited only the internalization step. The assay described here may be useful in studies concerned with the function and expression of phagocytosis-mediating surface lectins.

Animals↗
PubMed2003

Measuring distances in supported bilayers by fluorescence interference-contrast microscopy: polymer supports and SNARE proteins.

Fluorescence interference-contrast (FLIC) microscopy is a powerful new technique to measure vertical distances from reflective surfaces. A pattern of varying intensity is created by constructive and destructive interference of the incoming and reflected light at the surface of an oxidized silicon chip. Different levels of this pattern are probed by manufacturing silicon chips with terraces of oxide layers of different heights. Fluorescence collected from membranes that are deposited on these terraces is then used to measure the distance of the fluorescent probes from the silicon oxide surface. Here, we applied the method to measure the distance between supported lipid bilayers and the surface of oxidized silicon chips. For plain fluid phosphatidylcholine bilayers, this distance was 1.7 +/- 1.0 nm. The cleft distance was increased to 3.9 +/- 0.9 nm in bilayers that were supported on a 3400-Da polyethylene glycol cushion. This distance is close to the Flory distance (4.8 nm) that would be expected for a grafted random coil of this polymer. In a second application, the distance of a membrane-bound protein from the membrane surface was measured. The integral membrane protein syntaxin1A/SNAP25 (t-SNARE) was reconstituted into tethered polymer-supported bilayers. A soluble form of the green fluorescent protein/vesicle-associated membrane protein (GFP-VAMP) was bound to the reconstituted t-SNAREs. The distance of the GFP from the membrane surface was 16.5 +/- 2.8 nm, indicating an upright orientation of the rod-shaped t-SNARE/v-SNARE complex from the membrane surface.

Antigens, Surface↗
PubMed1997

Growth cone interactions with purified cell and substrate adhesion molecules visualized by interference reflection microscopy.

The migration of growth cones on substrates consisting of naturally occurring cell adhesion molecules has been extensively studied in cell culture. However, relatively little is known about how growth cones contact the substrate or how the patterns of contact change as growth cones move forward. We have examined the interactions of chick retinal ganglion cell growth cones with laminin, merosin, N-cadherin, L1 and poly-L-lysine by time-lapse interference reflection microscopy (IRM) using a laser scanning confocal microscope. In images obtained by IRM, areas of a cell that are closely apposed to the substrate appear dark whereas areas that are farther away appear light. Growth cones on Jaminin and merosin were almost uniformly light, indicating that very little of the membrane was in close contact with the substrate. Growth cones on N-cadherin had a mottled appearance with some relatively large dark gray areas. The proximal portions of filopodia often were dark, in contrast to those on laminin and merosin which were light. In addition, growth cones on N-cadherin had numerous dark gray punctate regions of close association with the substrate. Growth cones on L1 had darker regions than growth cones on other substrates and these comprised a larger fraction of their area. There also were differences in the temporal dynamics of growth cone interactions with different substrates and these differences correlated with differences in rates of growth. None of the contacts observed in growth cones were as dark or stable as focal contacts of fibroblasts.

Animals↗
PubMed1979

Sputum screening by Nomarski interference contrast microscopy.

Gram-stained smears of specimens submitted for sputum cultures were compared with direct wet mounts examined by Nomarski interference contrast microscopy (NIM) for enumeration of squamous epithelial cells (EPC) and leukocytes (WBC). The results obtained by the two methods were comparable, but specimens were more rapidly screened and cell types were more readily differentiated by NIM. Specimens submitted for sputum culture over a 3-month period were examined for EPC and WBC by NIM. Twenty-two percent of the specimens had greater than 25 EPC/field or a predominance of EPC (class I), 30% had greater than 25 EPC and greater than 25 WBC/field (class II), and 48% had greater than 25 WBC/field or a predominance of WBC (class III). The clinical relevance of the culture results was determined by reviewing the records of patients whose specimens were included in the study. Class I specimens provided only 30% clinically relevant culture results. Specimens in class II provided useful culture results in 63% of the patients, and 96% of those in class III provided clinically relevant information. The results confirm the value of sputum screening and demonstrate that NIM provides a rapid, simple, and accurate method for sputum screening.

Diagnosis, Differential↗
PubMed2002

Differential interference contrast microscopy as a polarimetric instrument.

Differential interference contrast (DIC) microscopy is shown to be equivalent to an incomplete Stokes polarimeter capable of probing optical properties of materials on microscopic-length scales. The Mueller matrix for a DIC microscope is calculated for various types of samples, and the polarimetric properties for DIC component parts of a spaceflight microscope are spectrally measured. As a practical application, a measurement of the index mismatch between colloidal particles and a nearly index-matched fluid bath was performed.

Journal Article↗
PubMed1998

Lipopolysaccharide-caused fragmentation of individual microtubules in vitro observed by video-enhanced differential interference contrast microscopy.

Microtubule disassembly is commonly believed to be a process of endwise tubulin dimer release. The present study demonstrates by video interference contrast microscopy that Escherichia coli lipopolysaccharide (LPS) caused microtubule disassembly in vitro by both endwise shortening and fragmentation. In contrast, the microtubules were only shortened from their ends in the presence of DNA, used as another example of a macromolecular microtubule effector. LPS-caused microtubule fragmentation was confirmed by transmission electron microscopy. Because of its ability to induce both fragmentation and endwise shortening, LPS, which is involved in sepsis pathogenesis, has to be regarded as a highly active microtubule-destabilizing agent.

Animals↗
PubMed2000

Trace formation during locomotion of L929 mouse fibroblasts continuously recorded by interference reflection microscopy (IRM).

The recently reported formation of highly ordered traces by migrating cells has been studied on L929 fibroblasts in time lapse experiments by means of interference reflection microscopy (IRM) as well as by conventional microscopy. Formation of pronounced traces on glass substrates correlates to migration after cell division, and the trace arrangement on the substrate depends on migration velocity: slow migration results in a highly branched, broad, and relatively short trace, while fast migration yields a slim and long trace with few branches. IRM-irradiation caused cessation of locomotion and trace formation and accelerated degradation of existing traces. Traces consist of cord-like cytoplasmic strands, which contain F-actin filaments and they seem to be enveloped by a membrane. It is supposed that cell traces are homologous to filopodia. Traces arise mainly from non-retracted filopodia at the rear margin of the migrating cell. The branches within the traces are the result of the repeated stretching out of a backwardly directed lamellipodium. They arise from the formation of new filopodia that emerge at the actin ribs of the lamellipodium.

Animals↗
PubMed2004

Linear phase imaging using differential interference contrast microscopy.

We propose an extension to Nomarski differential interference contrast microscopy that enables isotropic linear phase imaging. The method combines phase shifting, two directions of shear and Fourier-space integration using a modified spiral phase transform. We simulated the method using a phantom object with spatially varying amplitude and phase. Simulated results show good agreement between the final phase image and the object phase, and demonstrate resistance to imaging noise.

Fourier Analysis↗
PubMed1988

Mapping cell-glass contacts of Dictyostelium amoebae by total internal reflection aqueous fluorescence overcomes a basic ambiguity of interference reflection microscopy.

The widespread ability of eukaryotic cells to produce thin cytoplasmic sheets or lamellae 100-200 nm thick can give rise to uncertainties in the interpretation of interference reflection microscopy (IRM) images when cell-substratum topography is the key interest. If allowed to spread upon a poly-L-lysine-coated surface, Dictyostelium discoideum amoebae typically form ultrathin lamellae of approximately equal to 100 nm thickness by cytoplasmic retraction. Whereas the cell body is grey, the lamellae appear very dark under IRM optics. These dark areas could be misinterpreted as stemming from a closer cell-substratum apposition beneath the lamellae than the cell body. This ambiguity can be avoided if the technique of total internal reflection aqueous fluorescence (TIRAF) is used in conjunction with a high refractive index glass (n = 1.83) as substratum. Contributions to the image generated by thin cytoplasm and also variable cytoplasmic refractive index are thereby minimized due to the extremely short range of the 'illuminating' evanescent wave. From our comparative IRM and TIRAF study of the ultrathin lamellae of Dictyostelium amoebae it is concluded that the cell-glass gap is relatively uniform beneath the entire cell. We briefly discuss the sensitivity of several cell types to TIRAF, the generation of ultrathin lamellae and the nature of the cell-glass gap.

Cell Adhesion↗
PubMed1986

Stages in axon formation: observations of growth of Aplysia axons in culture using video-enhanced contrast-differential interference contrast microscopy.

The regenerative growth in culture of the axons of two giant identified neurons from the central nervous system of Aplysia californica was observed using video-enhanced contrast-differential interference contrast microscopy. This technique allowed the visualization in living cells of the membranous organelles of the growth cone. Elongation of axonal branches always occurred through the same sequence of events: A flat organelle-free veil protruded from the front of the growth cone, gradually filled with vesicles that entered by fast axonal transport and Brownian motion from the main body of the growth cone, became more voluminous and engorged with organelles (vesicles, mitochondria, and one or two large, irregular, refractile bodies), and, finally, assumed the cylindrical shape of the axon branch with the organelles predominantly moving by bidirectional fast axonal transport. The veil is thus the nascent axon. Because veils appear to be initially free of membranous organelles, addition of membrane to the plasmalemma by exocytosis is likely to occur in the main body of the growth cone rather than at the leading edge. Veils almost always formed with filopodial borders, protruding between either fully extended or growing filopodia. Therefore, one function of the filopodia is to direct elongation by demarcating the pathway along which axolemma flows. Models of axon growth in which the body of the growth cone is pulled forward, or in which advance of the leading edge is achieved by filopodial shortening or contraction against an adhesion to the substrate, are inconsistent with our observations. We suggest that, during the elongation phase of growth, filopodia may act as structural supports.

Animals↗
PubMed1989

Response of microbial adhesives and biofilm matrix polymers to chemical treatments as determined by interference reflection microscopy and light section microscopy.

The polymers involved in the adhesion of Pseudomonas fluorescens H2S to solid surfaces were investigated to determine whether differences between cell surface adhesives and biofilm matrix polymers could be detected. Two optical techniques, i.e., interference reflection microscopy (IRM) and light section microscopy (LSM), were used to compare the responses of the two types of polymer to treatment with electrolytes, dimethyl sulfoxide (DMSO), and Tween 20. To evaluate initial adhesive polymers, P. fluorescens H2S cells were allowed to attach to glass cover slip surfaces and were immediately examined with IRM, and their response to chemical solutions was tested. With IRM, changes in cell-substratum separation distance between 0 and ca. 100 nm are detectable as changes in relative light intensity of the image; a contraction of the polymer would be detected as a darkening of the image, whereas expansion would appear as image brightening. To evaluate the intercellular polymer matrix in biofilms, 3-day-old biofilms were exposed to similar solutions, and the resultant change in biofilm thickness was measured with LSM, which measures film thicknesses between 10 and 1,000 microns. The initial adhesive and biofilm polymers were similar in that both appeared to contract when treated with electrolytes and to expand when treated with Tween 20. However, with DMSO treatment, the initial adhesive polymer appeared to contract, whereas there was no change in thickness of the biofilm polymer. These results indicate that both polymers bear acidic groups and thus act electrostatically with cations and are able to enter into hydrophobic interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Adhesins, Bacterial↗
PubMed2004

Direct observation of single native DNA molecules in a microchannel by differential interference contrast microscopy.

Direct observation of single native DNA molecules in a microchannel was monitored without fluorescence-dye labeling. At a PDMS/glass microchip, the image of individual lambda-DNA molecules appear sharp and distinct in Nomarski differential interference contrast microscopy. Intercalator dyes affected the physical properties and dynamic behavior of individual DNA molecules. From the migration velocities in the microchannel it is evident that native DNA molecules migrated faster than DNA molecules labeled with the intercalator YOYO-1. This is because YOYO-1 increases the molecular weight and size of lambda-DNA and decreases the charge. The electric field strength and pH also affected the dynamics of single DNA molecules. We also observed that YOYO-labeled DNA was more stretched out compared to native DNA.

Benzoxazoles↗
PubMed1981

Eosinophil degranulation. Monitoring by interference contrast microscopy.

A method is described for the quantitative monitoring of human eosinophil degranulation using interference contrast microscopy. Using staphyloccoci as a stimulus, degranulated cells appeared larger than nondegranulating cells, were ameboid in shape and exhibited large nude areas of cytoplasm with prominent nuclei. Granules were observed to marginate along the plasma membrane and discharge into the exterior of the cell. Eosinophils that were not induced to degranulate were spherical in shape and the cytoplasm contained numerous granules that often obscured the nuclei. Pharmacological agents that increase intracellular cAMP prevented degranulation, whereas those that increase cGMP had no effect on degranulation. Colchicine inhibited degranulation but did not interfere with the phagocytosis of staphyloccoci. Endotoxin-activated serum, ECF-A, phytohemagglutinin, concanavalin A, levamisole, and compound 48/80 caused degranulation of eosinophils per se. The presence of disodium cromoglycate prevented this degranulation. Compound 48/80 and disodium cromoglycate had no effect on the level of intracellular cAMP and cGMP.

Chemotactic Factors↗
PubMed2006

Atypical mobilities of single native DNA molecules in microchip electrophoresis revealed by differential interference contrast microscopy.

A transmitted-light optical microscope using differential interference contrast (DIC) was employed to follow the real-time dynamics of different kb-sized single native dsDNA molecules without fluorescent-dye labeling. In a PDMS/glass microchip, the electrophoretic migration velocities of large dsDNA molecules are lower than small dsDNA molecules in a running buffer of 0.25% v/v nonionic polymeric surfactant C16E6 (n-alkyl polyoxyethylene ether) in 100 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffer because the C16E6 behaved as a dynamic polymer. However, the order of migration reversed in 50 mM Gly-Gly buffer. The radial positions of individual DNA molecules (i.e., center or walls of the microchip) did not change the migration order. The atypical migration order correlated well with the results in CE. The alignment of the deformable molecules due to viscous drag is likely responsible for these observations.

Buffers↗
PubMed1984

Morphologic response of the rabbit cortical collecting tubule to peritubular hypotonicity: quantitative examination with differential interference contrast microscopy.

The isolated and perfused cortical collecting tubule of the rabbit was examined by differential interference contrast microscopy in order to characterize the morphologic response of this nephron segment to peritubular hypotonicity. Computer-assisted, morphometric procedures were developed to obtain measurements of cell volume and lateral intercellular space geometry from interference contrast images of perfused nephron segments. Following dilution of the bath from 290 to 190 mOsm in the absence of antidiuretic hormone (T = 25 degrees C), the cells swelled rapidly to a new steady-state volume which was maintained for at least 20 to 30 min and which was about 90% of that predicted for ideal osmometric behavior. The increase in cell volume was accomplished entirely by bulging of the cells into the lumen; lateral space width and outside tubule diameter were unaffected by peritubular hypotonicity. In addition, the swelling of the cells was associated with an apparent swelling of intracellular organelles, e.g., nuclei and mitochondria. Our results indicate that cells of the mammalian collecting tubule swell without the capacity for significant volume regulation at 25 degrees C and without the cytoplasmic vacuolation and dilation of the lateral intercellular spaces observed following the onset of antidiuretic hormone-dependent volume reabsorption (E. Ganote , J. Grantham , H. Moses, M. Burg and J. Orloff , J. Cell Biol. 36:355, 1968).

Animals↗
PubMed1985

Interference reflection microscopy in cell biology: methodology and applications.

Since its introduction into cell biology by Curtis in 1964, interference reflection microscopy (IRM) has been used by an increasing number of researchers to study cell-substrate interactions in living cells in culture. With the use of antiflex objectives, high-contrast IRM images can now be readily obtained. From the different theories on image formation in IRM that have been put forward, it can be seen that a zero-order interference pattern is generated at high illuminating numerical aperture. This yields information on the closeness of contact between cell and substrate, with only minor perturbation by reflections from the dorsal cell surface. Therefore, the proper use of illuminating apertures is crucial. Nevertheless, IRM images have to be interpreted with caution, especially under thin cytoplasmic sheets. Quantitative IRM is possible only with a mathematical model for finite illuminating aperture interferometry and with an independent measurement of cell thickness for values up to 1 micron. IRM has been applied qualitatively to a large number of cell types, and it seems that there are two universal types of adhesion. Focal contacts are small regions of closest cell-substrate apposition, possibly of immediate contact, that are associated with the distal end of actin filament bundles. They are firm attachment structures that hold the cell in place and in its spread shape. Close contacts are broad areas of reduced cell-to-substrate distance. They are weaker but highly dynamic adhesions that sustain rapid movements of cells or cell parts over the substrate. Although a number of independent observations suggest that adhesion patterns of malignantly transformed cells differ from those of their normal counterparts, there is no simple correlation between malignancy in vivo and altered contact formation in vitro. The adhesion pattern seems to be determined by the locomotory state of the cells rather than by their tissue of origin. Finally, IRM can also be used to enhance contrast in images of fixed preparations.

Animals↗
PubMed1990

Visualization of living terminal hypertrophic chondrocytes of growth plate cartilage in situ by differential interference contrast microscopy and time-lapse cinematography.

The functional unit within the growth plate consists of a column of chondrocytes that passes through a sequence of phases including proliferation, hypertrophy, and death. It is important to our understanding of the biology of the growth plate to determine if distal hypertrophic cells are viable, highly differentiated cells with the potential of actively controlling terminal events of endochondral ossification prior to their death at the chondro-osseous junction. This study for the first time reports on the visualization of living hypertrophic chondrocytes in situ, including the terminal hypertrophic chondrocyte. Chondrocytes in growth plate explants are visualized using rectified differential interference contrast microscopy. We record and measure, using time-lapse cinematography, the rate of movement of subcellular organelles at the limit of resolution of this light microscopy system. Control experiments to assess viability of hypertrophic chondrocytes include coincubating organ cultures with the intravital dye fluorescein diacetate to assess the integrity of the plasma membrane and cytoplasmic esterases. In this system, all hypertrophic chondrocytes, including the very terminal chondrocyte, exist as rounded, fully hydrated cells. By the criteria of intravital dye staining and organelle movement, distal hypertrophic chondrocytes are identical to chondrocytes in the proliferative and early hypertrophic cell zones.

Animals↗
PubMed1995

Engineered fusion molecules at chelator lipid interfaces imaged by reflection interference contrast microscopy (RICM).

In molecular biology, biotechnology, and protein-engineering, the expression of histidine fusion proteins is a very powerful technique for the identification and one-step purification based on the interaction of the histidine stretch with immobilized metal complexes. By synthesis of a novel class of chelator lipids, this technique was combined with the concept of self-assembly leading to interfaces for immobilization and orientation of histidine-tagged biomolecules (Schmitt et al., 1994). Here, the chelator lipid layers were transferred onto solid substrate by vesicle fusion and Langmuir-Blodgett-techniques. Specific binding of a peptide containing an oligohistidine sequence to these functionalized interfaces was demonstrated by reflection interference contrast microscopy (RICM). Due to the phase separation behaviour of lipid mixtures, the chelator lipid interface could be further structured in two dimensions. Binding and organization of histidine-tagged molecules at these two-dimensional recognition arrays was imaged by RICM with a layer thickness resolution of 0.2 nm, and 0.5 microm laterally. Specific docking can be triggered by adding nickel ions and disrupted by EDTA. This concept opens up possibilities for reversible immobilization, enrichment and organization of histidine fusion proteins at interfaces and their application in biosensing.

Amino Acid Sequence↗