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PubMedDate not supplied

New methods for cytotoxicity testing: quantitative video microscopy of intracellular motion and mitochondria-specific fluorescence.

The aim of this study is to qualify the application the new microscopic methods fluorescence and AVEC-DIC (Allen video-enhanced contrast differential interference contrast) microscopy for toxicity testing. The effects of 2-OH-ethyl methacrylate (HEMA), a toxic acrylic monomer, on human fibroblasts was tested. The HEMA concentrations used were 0.01-1% at incubation times of 1-24 h. The cells were observed with AVEC-DIC microscopy and fluorescent staining to evaluate the velocity of lysosomal movement, the number and morphology of the mitochondria, and the fine structure of the cell. In the samples treated with the toxic compound the lysosomal movement changed, as did the morphology of the mitochondria and of the whole cells. The results are compared and discussed with regard to the results of conventional cytotoxicity tests performed in parallel. The new methods proved to be more sensitive and yielded more specific information on the cellular changes caused by the compound.

Cell Movement↗
PubMed1982

Reorganization of cytoskeletal and contractile elements during transition of human monocytes into adherent macrophages.

Indirect immunofluorescence microscopy was used to study the reorganization of contractile and cytoskeletal elements of human monocytes during their in vitro transition into macrophage-like cells. In freshly isolated monocytes, actin, myosin, alpha-actinin, and vinculin all had a diffuse distribution, whereas microtubules and vimentin-type intermediate filaments showed a distinct fibrillar organization. In culture, the cells differentiated within 1 week to two distinct cell types: bipolar fibroblastoid cells and flattened epithelioid cells. A conspicuous redistribution of actin, alpha-actinin, and vinculin from a diffuse cytoplasmic location into distinct punctate foci at the substratum-facing side of the cells was seen already at the early attachment phase of the spreading monocytes. In spread cells the punctate foci occupied the whole ventral surface of the cells, and in flattened, fully spread cells these proteins formed distinct punctate plaques at the under surface of the cells excluding however, the ruffle edge-like membrane regions. At the plaques, a close co-distribution of actin and vinculin was seen in double indirect immunofluorescence microscopy. Myosin showed a distinctly different reorganization during the transition process changing from a diffuse cytoplasmic location to a striated, surface-associated distribution. Interference reflection microscopy revealed large areas of close adhesion but no focal adhesion sites in spreading monocytes. Cytochalasin B treatment lead to a rapid distortion of the actin organization and resulted in clump-like cytoplasmic aggregates and nuclear paracrystals of actin concomitantly with the rounding up of the cells. Vimentin filaments were seen as a perinuclear aggregate until the acquisition of the fully spread morphology. At this stage both vimentin filaments and microtubules displayed a fibrillar appearance throughout the cytoplasmic domain. Treatment of the spread monocytes with antimitotic drugs caused a retraction of the cell from beyond the attachment plaques but had no effect on the punctate actin organization. The results show that in the monocyte-macrophage transition there is an extensive cytoskeletal reorganization that can be correlated with the different phases of the cell-to-substratum attachment process.

Actinin↗
PubMed1984

Bidirectional transport of fluorescently labeled vesicles introduced into extruded axoplasm of squid Loligo pealei.

A reconstituted model was devised to study the mechanisms of fast axonal transport in the squid Loligo pealei. Axonal vesicles were isolated from axoplasm of the giant axon and labeled with rhodamine-conjugated octadecanol, a membrane-specific fluorescent probe. The labeled vesicles were then injected into a fresh preparation of extruded axoplasm in which endogenous vesicle transport was occurring normally. The movement of the fluorescent, exogenous vesicles was observed by epifluorescence microscopy for as long as 5 min without significant photobleaching, and the transport of endogenous, nonfluorescent vesicles was monitored by video-enhanced differential interference-contrast microscopy. The transport of fluorescent, exogenous vesicles was shown to be bidirectional and ATP-dependent and occurred at a mean rate of 6.98 +/- 4.11 micron/s (mean +/- standard deviation, n = 41). In comparison, the mean rate of transport of nonfluorescent, endogenous vesicles in control axoplasm treated with vesicle buffer alone was 4.76 +/- 1.60 micron/s (n = 64). These rates are slightly higher than the mean rate of endogenous vesicle movement in extruded axoplasm (3.56 +/- 1.05 micron/s, n = 40) not subject to vesicles or vesicle buffer. Not all vesicles and organelles, exogenous or endogenous, were observed to move. In experiments in which proteins of the surface of the fluorescent vesicles were digested with trypsin before injection, no movement of the fluorescent vesicles was observed, although the transport of endogenous vesicles and organelles appeared to proceed normally. The results summarized above indicate that isolated vesicles, incorporated into axoplasm, move with the characteristics of fast axonal transport. Because the vesicles are fluorescent, they can be readily distinguished from nonfluorescent, endogenous vesicles. Moreover, this system permits vesicle characteristics to be experimentally manipulated, and therefore may prove valuable for the elucidation of the mechanisms of fast axonal transport.

Animals↗
PubMed1979

Cell surface lipid in the amniotic fluid.

Limitations of cell staining and light microscopy have led to confusion as to the etiology and morphologic characteristics of nile-blue-sulfate-stained orange cells in amniotic fluid. Nomarski Differential Interference Contrast Microscopy (NDICM) was used to study these cells as they appear in their unstained state. The presence of lipid-bearing cells was correlated with gestational age and mode of delivery, and incubation studies were applied toward an evaluation of the mechanism of lipid formation. Lipid-bearing cells in the amniotic fluid appeared after 37 1/2 weeks' gestation in association with fetal lung maturity.

Amniotic Fluid↗
PubMed1984

Relationship of heparan sulfate proteoglycans to the cytoskeleton and extracellular matrix of cultured fibroblasts.

The distribution of heparan sulfate proteoglycans (HSPG) on cultured fibroblasts was monitored using an antiserum raised against cell surface HSPG from rat liver. After seeding, HSPG was detected by immunofluorescence first on cell surfaces and later in fibrillar deposits of an extracellular matrix. Cell surface HSPG aligned with microfilament bundles of rat embryo fibroblasts seen by phase-contrast microscopy but was diffuse on transformed rat dermal fibroblasts (16C cells) which lack obvious stress fibers. Focal adhesions isolated from either cell type and monitored by interference reflection microscopy showed a concentration of HSPG labeling with respect to the rest of the membrane. Increased labeling in these areas was also seen for fibronectin (FN) by using an antiserum that detects both plasma and cell-derived FN. Double immunofluorescent staining of fully adherent rat embryo fibroblast cells showed some co-distribution of HSPG and FN, and this was confirmed by immunoelectron microscopy, which detected HSPG at localized areas of dorsal and ventral cell membranes, overlapping cell margins, and in the extracellular matrix. During cell shape changes on rounding and spreading, HSPG and FN may not co-distribute. Double labeling for actin and either HSPG or FN showed a closer correlation of actin with HSPG than with FN. The studies are consistent with HSPG being closely involved in a transmembrane cytoskeletal-matrix interaction; the possibility that HSPG coordinates the deposition of FN and other matrix components with cytoskeletal organization is discussed.

Animals↗
PubMed1999

Endothelial gaps as sites for plasma leakage in inflammation.

OBJECTIVE: In 1961, Majno and Palade proposed that plasma leakage in acute inflammation caused by histamine, serotonin, or bradykinin results via gaps that form between endothelial cells of postcapillary venules. Now the relevance of endothelial gaps in plasma leakage is being questioned. The purpose of this review is to summarize experimental evidence from our studies showing that endothelial gaps participate in plasma leakage in inflammation. METHODS: Using neurogenic inflammation as a model of plasma leakage in acute inflammation, we compared five methods to determine whether endothelial gaps form in the microvasculature of the rat trachea. 1) Endothelial cells borders and gaps were stained with silver nitrate and visualized by light, scanning, and transmission electron microscopy. 2) The luminal surface of endothelial cells was examined by scanning electron microscopy. 3) The luminal surface of endothelial cells was stained with a biotinylated lectin and avidin-biotin-peroxidase histochemistry, and then was examined by differential interference contrast microscopy. 4) Endothelial junctions were reconstructed from serial sections photographed by transmission electron microscopy. 5) Leakage was measured after perfusion of lectins or tracers through aldehyde-fixed vessels in situ. RESULTS: The results from the five methods used in this system were consistent with the formation of gaps between endothelial cells. Endothelial gaps were rare or absent under baseline conditions, but appeared with the onset of plasma leakage and had a distribution that matched the distribution of leakage. Gaps had a complex morphology and were accompanied by fingerlike cell processes, which may anchor adjacent endothelial cells to one another and participate in gap closure. In contrast to normal vessels, vessels that were leaky in life continued to leak after aldehyde fixation, in evidence that, once formed, the leakage pathway did not require energy-dependent membrane movement or vesicle shuttling. Holes through endothelial cells were less than 1% as frequent as intercellular gaps. CONCLUSIONS: Taken together, the results show that endothelial gaps are a consistent feature of leaky vessels in the model system we studied, and are not an artifact of a particular method. The morphological complexity of the openings and accompanying fingerlike cell processes and overlapping endothelial cell borders make gaps difficult to distinguish from transcellular holes in thin sections viewed by transmission electron microscopy. However, scanning electron microscopic observations show that most of the openings in leaky venules are intercellular gaps, not transcellular holes. The formation and closure of gaps are likely to be energy-dependent, but the process of plasma leakage is not, provided there is adequate driving force for extravasation. The cellular mechanisms of gap opening and closure remain to be elucidated.

Animals↗
PubMedDate not supplied

Differential polarization microscopy of changes in structure in spermatocyte nuclei.

Phase-dependent forms of microscopy (such as phase contrast, interference, or polarization microscopy) have been used for many years; the amount of phase retardation produces images based mainly on index of refraction. We have developed a microscope that forms images that depend on small differences in extinction for different forms of incident polarized light. By modulating the polarization of light incident on the sample and digitally recording the difference in intensities of transmitted light, we obtain images which specifically reveal either ordered linear structures or chiral (right- or left-handed) structures. Linearly polarized light, incident alternately with two perpendicular directions of polarization, forms images of structures which have linear order or linear orientation. Right-handed and left-handed circularly polarized light incident alternately on a sample forms images of chiral structures. Structures with neither linear order nor chirality are essentially invisible. Thus, images based on linear dichroism, circular dichroism, and linear and circular differential scattering can be used to detect specific types of structures which may be difficult to observe by conventional methods. We have used such 'linear and circular differential imaging' to study the structure of the nucleolus (the site of RNA synthesis) in live primary spermatocytes of Drosophila when they are transcriptionally active or inactive. Some inactive nucleoli are bipartite, with two distinct structures visible by differential scattering of both linearly and circularly polarized light. The active nucleolus is a single domain; it is clearly distinguished from part of the Y chromosome, and it shows different internal structure with linearly and circularly polarized light. Thus, polarization-dependent images reveal structures which can be associated with the transcriptional activity of cells.

Animals↗
PubMed1978

The observation of collagen and elastin structures in wet whole mounts of pulmonary and aortic leaflets.

Porcine pulmonary and aortic heart valve leaflets have been examined as wet whole mounts with the Nomarski differential-contrast imaging technique. The fibrous components, collagen and elastin, which comprise the load-bearing components of the tissue are imaged readily as a function of location on the leaflets as well as of depth through their section. The response of the collagen and elastin structures to applied stress was observed directly with a simple straining device fitted to the microscope stage. I believe that the method has considerable advantages over standard histologic or scanning electron microscope techniques in that detailed structural studies can be related easily to the gross leaflet structure and that there is no interference with the biologic properties of the tissue.

Animals↗
PubMed2005

Diffraction-induced coherence levels.

We examined the influence of complex diffraction effects on low-coherence fringes created for high-aspect depth-to-width ratio structures called trenches. The coherence function was analyzed for these micrometer-wide trenches and was registered with a white-light interference microscope. For some types of surface structure we observed that additional low-coherence fringes that do not correspond directly to the surface topology are formed near the sharp edges of the structures. These additional coherence fringes were studied by rigorous numerical evaluations of vector diffractions, and these simulated interference fields were then compared with experimental results that were obtained with a white-light interference microscope.

Computer Simulation↗
PubMed1990

Echistatin is a potent inhibitor of bone resorption in culture.

The venom protein, s-echistatin, originally derived from the saw-scaled viper Echis carinatus, was found to be a potent inhibitor of bone resorption by isolated osteoclasts. This Arg24-Gly25-Asp26-(RGD)-containing protein inhibited the excavation of bone slices by rat osteoclasts (IC50 = 0.1 nM). It also inhibited the release of [3H]proline from labeled bone particles by chicken osteoclasts (IC50 = 100 nM). By comparison, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibited resorption by rat or chicken osteoclasts with an IC50 of 0.1 mM while ala24-echistatin was inactive. Video microscopy showed that rat osteoclast attachment to substrate was more sensitive to s-echistatin than was the attachment of mononuclear cells or chicken osteoclasts. The difference in sensitivity of rat and chicken osteoclasts to s-echistatin may be due to differences between receptors on rat and chicken osteoclasts for s-echistatin. Antibody localization of echistatin on these cells showed much greater echistatin binding to rat osteoclasts than to chicken osteoclasts. Laser scanning confocal microscopy after immunohistochemical staining showed that s-echistatin binds to osteoclasts, that s-echistatin receptors are most abundant at the osteoclast/glass interface, and that s-echistatin colocalizes with vinculin. Confocal interference reflection microscopy of osteoclasts incubated with s-echistatin, demonstrated colocalization of s-echistatin with the outer edges of clusters of grey contacts at the tips of some lamellipodia. Identification of the echistatin receptor as an integrin was confirmed by colocalization of echistatin fluorescence with staining for an alpha-like subunit. Attachment of bone particles labeled with [3H]proline to chicken osteoclasts confirmed that the mechanism of action of echistatin was to inhibit osteoclast binding to bone presumably by disrupting adhesion structures. These data demonstrate that osteoclasts bind to bone via an RGD-sequence as an obligatory step in bone resorption, that this RGD-binding integrin is at adhesion structures, and that it colocalizes with vinculin and has an alpha-like subunit.

Amino Acid Sequence↗