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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↗

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↗

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↗

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↗

PubMed2000

Rotational-diversity phase estimation from differential-interference-contrast microscopy images.

An iterative phase-estimation method for the calculation of a specimen's phase function or optical-path-length (OPL) distribution from differential-interference-contrast (DIC) microscopy images is presented. The method minimizes the least-squares discrepancy measure by use of the conjugate-gradient technique to estimate the phase function from multiple DIC images acquired at different specimen rotations. The estimate is regularized with a quadratic smoothness penalty. Results from testing the method with simulations and measured DIC images show improvement in the estimated phase when at least two rotationally diverse DIC images instead of a single DIC image are used for the estimation. The OPL of a cell that is estimated from two DIC images was found to be much more reliable than the OPL computed from single DIC images (which had a coefficient of variation equal to 15.8%).

Computer Simulation↗

PubMed1992

Observations on the turkey oviductal sperm-storage tubule using differential interference contrast microscopy.

Squash preparations of unfixed, uterovaginal junction mucosae revealed that openings to sperm-storage tubules were round or slit-like and were surrounded by either cilia, which were part of the uterovaginal junction surface epithelium, or nonciliated cells resembling the sperm-storage tubule epithelium. By focusing on different levels of the sperm-storage tubule (optical sectioning), connective tissue fibres and cells between individual sperm-storage tubules, epithelium and lumen of sperm-storage tubules containing resident spermatozoa were observed. An optical section through the sperm-storage tubule epithelium revealed basal nuclei and associated nucleoli, and refractile supranuclear lipid droplets. Luminal spermatozoa were distributed primarily in the distal third of the sperm-storage tubule and nearly always formed a tight bundle at its base. These spermatozoa were often observed slowly and synchronously oscillating. In two-thirds of the 30-week-old, non-photostimulated hens, sperm-storage tubules were fully formed. In contrast, the remaining hens possessed bud-like surface invaginations lacking discernible lumina. It was concluded that differential interference contrast microscopy offers better spatial and optical resolution of the sperm-storage tubule than other modes of light microscopy.

Animals↗

PubMed1976

Observations of the microcirculatory bed in rat mesocecum using differential interference constrast microscopy in vivo and electron microscopy.

The microvascular bed of the rat mesocecum has been examined in vivo using differential interference (Nomarski) optics and subsequently by electron microscopy. The preferential channel, from terminal arteriole to collecting venule, has been examined. In the terminal arteriolar segment the endothelial layer is covered by a continuous layer of smooth muscle cells which, in turn, are surrounded by adventitia. In the metarteriolar segment the periendothelial cells still resemble smooth muscle cells but the tunica media is discontinuous. In the distal segment periendothelial cells are more scattered and have the appearance of pericytes. There appears to be a continuous transition of the periendothelial cell layer from terminal arteriole to distal segment. Nerve endings were seeen in both the terminal arteriolar and metarteriolar segments. During contraction smooth muscle cells, oriented circumferentially, shorten and thicken. Endothelial cells appear anchored by myoendothelial junctions. Endothelial cells have filaments which show increased banding during vasoconstriction, suggesting that such cells may contract. Capillary offshoots leave the preferential channel, usually at right angles. Smooth muscle cells are oriented to form a sphincter and there are many myoendothelial junctions at the branch point. Within a short distance the capillary branch loses its periendothelial coat.

Animals↗

PubMed1983

Motility of human polymorphonuclear neutrophils: microscopic analysis of substrate adhesion and distribution of F-actin.

Directed movement of polymorphonuclear neutrophils (PMN) requires cell polarization and the orderly making and breaking of cell-substrate contacts. We compared the movement of human PMN suspended from the underside of glass coverslips to that of PMN seen in "profile" on fibers, using brightfield, differential interference contrast and reflection interference microscopy. Images were recorded on film and videotape and analyzed in real time and time lapse. The distribution of F-actin was observed with image-enhanced fluorescence microscopy after staining with NBD-phallacidin. PMN exhibited two patterns of motility. Fifteen to twenty-five percent of cells moved in a low profile gliding pattern and exhibited caudad displacement of dorsal surface folds. Most PMN made progress by cycles of partial release of the lamellipodium from the substrate and anterior advance followed by arching or rolling and lamellipodial reassociation with the substrate. Cells stimulated with bacteria, casein, or chemotactic formyl peptide rarely spread on the coverglass but waved into the medium attached only by the uropod. Eventually, many detached completely from the substrate. Cells confined to the substrate surface with overlying agarose were able to locomote when confronted with these substances. F-actin was irregularly distributed in nonpolarized suspended cells but concentrated in the lamellipodium in polarized cells. As cells arched along a substrate, F-actin accumulated in foci corresponding to the substrate-PMN interface, particularly at the uropod and retraction fibrils. Conversely, cells that were physically restricted to movement in the plane of the substrate surface by overlying agarose exhibited diffuse F-actin along the entire cell. Suspended PMN polarized with formyl peptide and incubated with Con A accumulated F-actin at the uropod. These observations suggest that both PMN locomotion and the movement of Con A binding sites involve the caudad redistribution of F-actin.

Actins↗

PubMed1990

Macrophages form circular zones of very close apposition to IgG-coated surfaces.

When phagocytes spread on surfaces coated with ligands such as IgG, they form a tight seal with the substrate. This seal excludes soluble macromolecules in the medium from the interface between the cell and substrate. In contrast, when cells spread on control surfaces that are not coated with ligands, the underside of the cell remains freely accessible to soluble proteins (Wright and Silverstein: Nature 309:359, 1984). We employed reflection-interference microscopy (RIM) to determine where the seal forms during interaction with ligand (IgG)-coated surfaces. Human monocyte-derived macrophages (MO) were plated at 37 degrees C on dinitrophenylated (DNP)-glass coverslips (control substrate), IgM anti-DNP-DNP-coated glass (control substrate), or on IgG anti-DNP-DNP-coated glass (phagocytosis-promoting substrate). Live or fixed cells were examined by RIM. Spreading on control surfaces at 37 degrees C was complete in 25 minutes, whereas spreading on IgG-coated surfaces was maximal within 15 minutes and resulted in cell-substrate contact area 1.6 X that of control cells. Within 1 h at 37 degrees C, 90% of MO that spread on IgG-coated substrates, but not on control substrates, excluded macromolecules from their underside. A minor population of cells (19%) exhibited a uniform iron gray RIM appearance indicating an even, close approach to the substrate. These cells may represent early stages of frustrated phagocytosis. In contrast to cells on control substrates, 70% of cells on IgG-coated substrates developed continuous peripheral dark rings in RIM indicative of close association with the substrate. Essentially all cells with peripheral dark rings in RIM excluded macromolecules from their underside. Enclosed within this ring was an area of greater separation between the cell membrane and the substrate, as indicated by the lighter grey of this region in RIM and by the accessibility of substrate to anti-substrate antibody when breaks in the dark ring occur. Thus, MO can create a closed compartment between plasma membrane and substrate that excludes proteins in the surrounding medium, thereby protecting substances secreted into this space from potentially inhibitory substances in the medium.

Cell Communication↗

PubMed1992

Fertilization alters the orientation of pigment granule saltations in Arbacia eggs.

Unfertilized eggs of the sea urchin Arbacia punctulata contain pigment granules distributed throughout their cytoplasm. During the first 15 minutes after fertilization, these vesicles move out to the cortex where they become firmly anchored. We have used time-lapse video differential interference microscopy to analyze the motility of these organelles in unfertilized and fertilized Arbacia eggs. Pigment granules exhibit saltatory movement in both unfertilized and fertilized eggs. Quantitation of vesicle saltations before and after fertilization demonstrates that while there is no significant difference in the speed or path-length of vesicle movement, there is a dramatic change in the orientation of these saltations. Saltations in the unfertilized egg are very non-radial and are as likely to be directed toward the cortex as away. In contrast, saltations in the fertilized egg are more radially oriented and more likely to be cortically directed. This transition must reflect underlying changes in the cellular structures necessary for pigment granule saltations. The change in the orientation of pigment granule saltations following fertilization requires both a transient increase in the cytoplasmic concentration of Ca2+ and an elevation of cytoplasmic pH. Similarly, the ability of pigment granules to adhere to the cortex requires both the transient elevation of cytoplasmic Ca2+ and the alkalinization of the cytoplasm. As the reorganization of cortical actin at fertilization is regulated by these ionic fluxes, and both movement and adhesion are sensitive to cytochalasins, we hypothesize that the alterations in directed motility and adhesion reflect underlying changes in the actin cytoskeleton.

Animals↗

PubMed1980

Changes in nucleolar dry mass of neurones of the paraventricular and supraoptic nuclei in the rat during pregnancy and lactation.

Interference microscopy was used to measure the dry mass of nucleoli in unfixed nuclei isolated from neurones of the paraventricular (PV) and supraoptic (SO) nuclei of female rats. Changes in nucleolar dry mass during pregnancy and lactation have been interpreted as reflecting changes in rates of synthesis of ribosomes and protein in these neurones. Measurements were made on a total of 6580 nucleoli from 135 rats. At the end of pregnancy nucleolar dry mass of both PV and SO neurones was increased compared with virgin female rats. Nucleolar dry mass of PV neurones but not SO neurones increased further during lactation. This change was biphasic, with a nadir at 2 weeks post partum. After day 5 post partum, nucleolar dry mass of PV and SO neurones was increased only in rats sucking pups. Adjustment of litter size to 10 or 22 to 24 pups on the first day post partum did not affect nucleolar changes in PV and SO neurones. Nucleolar changes were less when only one pup was nursed. The results are discussed in relation to oxytocin secretion induced by the suckling stimulus and the synthetic response of PV and SO neruones to increased secretion.

Animals↗