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At least 379 records · Page 21Linked to original sources
PubMed2004

Novel method for quantification of brain cell swelling in rat hippocampal slices.

We have developed a novel device for the quantification of edematous morphology changes in acute brain slices. We can also carry out real-time monitoring of detailed hippocampal cells. The device we developed is based on infrared differential interference contrast microscopy (IR-DIC) and a custom-made real-time computerized image-analysis system for quantification of the morphological dynamics of cells in slice preparations. We applied the coefficient of variation (CV) of light intensity in IR-DIC images to evaluate the change in morphological dynamics. We examined three kinds of edema in the CA1 region of rat hippocampal acute slices under conditions of hypotonic, strong excitation, and experimental ischemia, together with field excitatory postsynaptic potential (fEPSP) recording from radiatum in CA1 the region. There were notable close relationships among the edema formations, the light transmittance, the extent of changes in CV, and features of fEPSP during the three different insults. The present results indicate that CV is a reliable quantification index for edema formation in brain tissue and confirm that applying CV for the analysis in addition to the light transmittance analysis presents additional important information on brain tissue swelling.

Animals↗
PubMed1985

Altered orientation of glycosaminoglycans and cellular changes in the tibial cartilage in the first two weeks of experimental canine osteoarthritis.

Changes in the cellularity and in the nature of the matrix were studied in the cartilages of the tibial plateau in experimentally induced arthritis in the dog, 7 and 14 days after section of the anterior cruciate ligament. Samples from the different regions of these cartilages were chilled and sectioned in a cryostat, with a variable microtome chuck to allow precise orientation of the specimens. The samples were examined by normal light microscopy, by microscopic interferometry, and by quantitative polarized light microscopy. The orientation of the glycosaminoglycans was assessed by the new "induced birefringence" method. The results indicated that only the region of the medial tibial cartilage that was unprotected by the meniscus was affected, showing increased water content, loss of superficial cells, and a decrease in orientation of the glycosaminoglycans. Whereas the birefringence of the collagen was unaffected, the superficial area that lacked oriented glycosaminoglycans was markedly increased; this may be a useful indicator of early osteoarthritic changes.

Alcian Blue↗
PubMed2005

Adhesion contact dynamics of fibroblasts on biomacromolecular surfaces.

Biomacromolecules like gelatin and chitosan have emerged as highly versatile biomimetic coatings for applications in tissue engineering. The elucidation of the interfacial kinetics of cell adhesion on biomacromolecular surfaces will pave the way for the rational design of chitosan/gelatin-based systems for cell regeneration. Biomacromolecular ultra-thin films, chemically immobilized on fused silica are ideal experimental models for determining the effect of surface properties on the biophysical cascades following cell seeding. In this study, confocal reflectance interference contrast microscopy (C-RICM), in conjunction with phase contrast microscopy and fluorescence confocal microscopy, was applied to detect the adhesion contact dynamics of 3T3 fibroblasts on chitosan and gelatin ultrathin films. X-ray photoelectron spectroscopy (XPS) confirmed the immobilization of chitosan or gelatin on the silanized glass surface. Both the initial cell deformation rate and the change of two-dimensional spread area of the 3T3 fibroblasts are higher on gelatin-modified surfaces than on chitosan surfaces. The steady-state adhesion energy of 3T3 fibroblasts on gelatin film is three times higher than that on chitosan film. Immuno-staining of actin further demonstrates the different organization of cytoskeleton, likely induced by the change in cell signaling mechanism on the two biomacromolecular surfaces. The better attachment of 3T3 fibroblast to gelatin is postulated to be caused by the presence of adhesive domains on gelatin.

3T3 Cells↗
PubMed1983

Histopathological diagnosis of hydatidiform mole.

Two microscopical methods are described which are useful for the identification of hydatidiform molar trophoblast. The first is an application of Nomarski's differential interference contrast microscopy which allows whole-mounted villi to be studied by optical sectioning. The second is an application of indirect immunofluorescence microscopy using a specific antiserum directed against the endothelial cell structural protein-known as vimentin.

Female↗
PubMedDate not supplied

Dynamic three-dimensional visualization of collagen matrix remodeling and cytoskeletal organization in living corneal fibroblasts.

The remodeling of extracellular matrices by cells plays a defining role in developmental morphogenesis and wound healing, as well as in tissue engineering. Three-dimensional (3-D) type I collagen matrices have been used extensively as an in vitro model for studying cell-induced matrix reorganization at the macroscopic level. However, few studies have directly assessed the dynamic process of 3-D matrix remodeling at the cellular and subcellular level. We recently developed an experimental model for investigating cell-matrix mechanical interactions by plating green fluorescen protein (GFP)-zyxin transfected cells inside fibrillar collagen matrices and performing high-magnification time-lapse differential interference microscopy (DIC) and wide-field fluorescent imaging. In this study, we extend this experimental model by performing four-dimensional (4-D) reflected light and fluorescent confocal imaging (using either visible light or multiphoton excitation) of living corneal fibroblasts transfected to express GFP-zyxin or GFP-alpha-actinin, 18 h after plating inside 3-D collagen matrices. Reflected light confocal imaging allowed detailed visualization of the cells and the fibrillar collagen surrounding them. By overlaying maximum intensity projections of reflected light and GFP-zyxin or GFP-alpha-actinin images and generating stereo pair reconstructions, 3-D interactions between focal adhesions and collagen fibrils in living cells could be visualized directly. Focal adhesions were generally oriented parallel to the direction of collagen fibril alignment in front of the cell. Killing the cells induced relaxation of transient cell-induced tension on the matrix; however, significant permanent remodeling always remained. Time-lapse 3-D imaging demonstrated an active response to the Rho-kinase inhibitor Y-27632, as indicated by cell elongation, extracellular matrix relaxation, and extension of pseudopodial processes. It is interesting that, at higher cell densities, groups of collagen fibrils were compacted and aligned into straps between neighboring cells. Overall, the continued development and application of this new approach should provide important insights into the basic underlying biochemical and biomechanical regulatory mechanisms controlling matrix remodeling by corneal fibroblasts.

Animals↗
PubMed2002

Systematic analysis of sporulation phenotypes in 624 non-lethal homozygous deletion strains of Saccharomyces cerevisiae.

A new high throughput mutant screening procedure for the detection of sporulation mutants was developed and used to analyse a set of 624 non-lethal homozygous deletion mutants created in the European joint research program EUROFAN. The screening procedure involved determination of LL- and DL-dityrosine, sporulation-specific compounds, which were shown to be robust markers of the extent and arrest stage of sporulation mutants. Secondary screens consisted of light microscopy to detect mature and immature spores and DAPI staining to monitor the progress of meiotic nuclear divisions. We discovered new phenotypic classes of mutants defective in spore wall synthesis that were not discovered by previous screens for sporulation mutants. The genes corresponding to the sporulation mutants fell in several functional classes, some of which were previously unknown to be involved in spore formation. Peroxisomes seem to play a role in spore wall synthesis. Mitochondria play a role in sporulation that is not simply restricted to supply of ATP from respiratory metabolism. The deletion mutants included in the set were functionally unknown at the start of EUROFAN; however, within the last few years the importance to sporulation of some of them was also reported by other authors. Taken together, about 8% of all single gene deletion mutants of non-essential genes of Saccharomyces cerevisiae seem to display a clear and reproducible sporulation phenotype.

Fluorescent Dyes↗
PubMed1996

Directed confrontations between fibroblasts and epithelial cells on micromachined grooved substrata.

Many aspects of cell social behavior, including aspects of tumor invasiveness, embryonic development, and wound healing, can be explained by the principle of contact inhibition (CI) of cell movement. CI refers to the tendency of fibroblasts cultured on a plane substratum to cease movement on contacting other fibroblasts. A problem in studying collisions between cells on a flat substratum is that it is difficult to control the specific regions of the cell that come in contact. In this study we used grooved micromachined titanium substrata to produce collisions between the following cell combinations: fibroblast/fibroblast, fibroblast/epithelium, and epithelium/epithelium. The cells were oriented by the substratum so that the leading lamellae of the cells confronted each other. Cell behaviors before and after contact were observed and recorded using time-lapse cinemicrography employing Nomarski reflected light differential interference microscopy. Electron-microscopy sections were prepared from areas where cell interactions occurred. Fibroblasts (F) moved significantly faster and more persistently on grooved than on smooth surfaces, but the speed of epithelial (E)-cell locomotion was not significantly altered. The grooves, however, guided the direction of locomotion for both cell types. When cultured on grooved surfaces in such a manner that the F and E cells collided head-on, the F, but not the E cells, frequently demonstrated contact inhibition of movement. However, after such collisions, significantly more F continued to invade the E sheet than were observed after F-E collisions on smooth surfaces. After F-F collisions on grooved surfaces, most cells moved to the sides of the grooves and continued in their original directions, while on smooth surfaces they moved off in various different directions. A possible explanation of these observations is that a grooved surface produces and maintains F polarity so that the direction of locomotion is less readily altered by cell-cell interactions.

Animals↗
PubMed1997

Reassembly of functional nucleoli following in situ unraveling by low-ionic-strength treatment of cultured mammalian cells.

In order to determine the most persistent components of the nucleolus that might serve as "core" nucleolar elements, we studied the reactivity of nucleoli in living mammalian cells subjected to hypotonic buffer saline followed by the incubation of the cells in an isotonic medium. To document as precisely as possible the fine structural changes which occurred, the cells were examined by video-enhanced optical microscopy, fluorescence confocal laser scanning microscopy, and electron microscopy combined with cytochemistry. Light microscopic autoradiography was used to demonstrate the transcriptional characteristics of the reassembled nucleoli. It was shown that all the major compartments of the intact nucleolus could be substantially affected by reduction of the osmolarity of the environmental media. The dynamic events of the nucleolar unraveling in low-salt buffers occurred in the following order: dispersion of the nucleolar pars granulosa, disassociation of the fibrillar complexes into discrete fibrillar centers (FCs) and the dense fibrillar component (DFC), and the almost complete unraveling of the DFC and FCs. At the terminal stages of nucleolar dispersion, the nuclear interior was mainly composed of a loose filamentous meshwork, and none of the typically discerned nucleolar constituents was recognized. Nevertheless, when hypotonically treated cells were returned to isotonic conditions, the nucleolar bodies rapidly began to reassemble. Within 1-2 h of cell incubation under isotonicity, the nucleoli not only became clearly visible, but also reconstituted to their initial size, shape, and position within the nucleus. The ultrastructure and functional activity of the reassembled nucleoli were also found to be fully comparable to those of the untreated controls. These data indicate that the architectural composition of the interphase nucleolus is strictly controlled by the cell. As far as could be determined, none of the usual substructures of the intact nucleolus that could be substituted by complete reassembly of the nucleolar bodies in normotonic conditions, including FCs and the DFC, remained clearly preserved in the terminal stage of nucleolar unraveling. We concluded that the integrity of the nucleolus was mainly preserved by the nuclear or nucleolar matrix system rather than by any other nucleolar structural domains.

Animals↗
PubMed1993

Pathfinding by neuroblastoma cells in culture is directed by preferential adhesion to positively charged surfaces.

Pathfinding is a fundamental behavior of migrating neuroblasts and advancing growth cones. We have analyzed this behavior in culture using mouse neuroblastoma (N1E-115) cells grown on a chemically patterned surface. The patterned surface was defined photolithographically and consisted of intersection 10-micron-wide pathways. The pathways were coated with positively charged amines and separated by regions bound with uncharged alkanes. Cells and growth cones were guided along the pathways and made choices at intersections. Whereas migrating cells made random choices at intersections, growth cones displayed a preference for advancing straight ahead. Interference reflection microscopy (IRM) revealed that pathfinding by cells and growth cones was correlated with greater overall attachment to aminated regions, although cell bodies and appendages also attached to adjacent alkanated regions. Thus guidance was not simply due to contact inhibition by alkanes; rather, it was due to "preferential" adhesion to aminated surfaces. Gray level analysis of IRM images demonstrated that focal and close contacts were made on both surfaces, indicating that preferential adhesion was not the result of tighter attachment to aminated surfaces. Fluorescent labeling of F-actin and microtubules indicated that preferential adhesion was not due to compartmentalization of these cytoskeletal structures on aminated regions. We propose that preferential adhesion involved a signal transduction mechanism that discriminated between positively charged and uncharged molecules. Such a mechanism could contribute to pathfinding by neuroblasts and growth cones along extracellular matrix proteins in vivo.

Acid-Base Equilibrium↗
PubMed1994

An experimental model for the exposure of human ciliated cells to sulfur dioxide at different concentrations.

Mucociliary transport is an important nonimmunological defense mechanism of the respiratory tract. The aim of this study was to investigate the effect of sulfur dioxide (SO2) at different concentrations on ciliary beat frequency (CBF). Ciliated cells were obtained from 12 volunteers by nose brush. CBF was quantified using video-interference microscopy. The cells were placed on a polycarbonate membrane in contact with the surface of a reservoir filled with RPMI 1640 (bicarbonate buffered) or Ringer's (electrolyte) solution, allowing the cells to be supplied by capillarity. In an exposure chamber the cells were exposed for 30 min to SO2 2.5-12.5 ppm at 37 degrees C and 100% air humidity. SO2 induced a dose-dependent decrease in CBF of the cells cultured in Ringer's solution. SO2 at 2.5 ppm caused a 42.8% decrease and at 12.5 ppm a 96.5% decrease (8.1 +/- 0.24 versus 0.28 +/- 0.20 Hz). CBF of cells cultured in RPMI 1640 was reduced only moderately after 12.5 ppm SO2 exposure (7.9 +/- 0.26 versus 6.70 +/- 0.30 Hz). In Ringer's solution a decrease in pH was observed after 30 min of SO2 exposure to 12.5 ppm to a minimum value of 3.6. By contrast, the pH of RPMI 1640 remained constant at 7.5 under identical conditions. After adding RPMI 1640 to Ringer's solution, CBF increased in parallel to the pH to control values (5.0 ppm: 4.64 +/- 0.45 to 8.51 +/- 0.60 Hz). These data suggest that the highly water-soluble SO2 reversibly eliminates CBF in correlation with a decrease in pH.

Adult↗
PubMed1990

Dioptrics of the facet lenses of male blowflies Calliphora and Chrysomyia.

1. The dioptrics of the facet lenses of two blowfly species, Calliphora erythrocephala and Chrysomyia megacephala, was investigated. Measurements were performed on facet lenses ranging in diameter from 20 to 80 microns. 2. The radius of curvature of the front surface of the facet lenses, measured by microreflectometry, increases approximately linearly with the facet lens diameter. 3. The optical path difference of the facet lens and water, measured by interference microscopy, depends on the distance from the optical axis according to a parabolic function. Average refractive index values, calculated from the optical path difference profile together with estimates of the thickness profile, are between 1.40 and 1.43, with the lowest values in the largest lenses. 4. The F-number calculated from the experimental data ranges from 1.5 to 2.2. It is argued that the range of effective F-numbers is 2.1-2.4.

Animals↗
PubMed1986

Cell shape and motility of oligodendrocytes cultured without neurons.

Oligodendrocytes, the myelin-forming cells of the central nervous system (CNS), were cultured from newborn rat brain and optic nerve to study how they differentiate in vitro in the absence of neurons. By use of galactocerebroside (GC) as a reference marker, the development of the cell phenotype was studied with video-enhanced differential interference contrast microscopy, immunofluorescence and electron microscopy. After a few days in culture, oligodendrocytes extend 5 to 10 main processes that are very rich in microtubules, but they did not stain with a monoclonal antibody reacting with all known classes of intermediate filaments. The number of processes can vary with the substrate on which the cells are grown; fewer processes form on laminin than on polylysine coated glass. Oligodendrocytes, in a fashion similar to that of neurons appear to keep their body immobile while the long processes grow. However, while neurons display motile activities mostly at the end of the cell processes called growth cones, the oligodendrocytes display motile, actin rich filopodia and lamellipodia along the entire length of all processes. The outgrowth of motile processes from oligodendrocytes sometimes occurs preferentially towards neighboring astrocytes. Oligodendrocyte processes display intense bidirectional movement of cytoplasmic organelles. Movement of surface components also occurs since GC molecules cross-linked by antibodies move from the processes towards the cell body. Thus, oligodendrocytes cultured without neurons develop on schedule a complex phenotype similar to their in vivo counterpart. In addition, their processes are capable of specific motile activities which may function in vivo to find the target axon and to transport myelin membrane components at the site of myelin assembly.

Animals↗
PubMed1980

Nomarski-optical studies of human chromosomes R-banded with barium hydroxide.

The morphologic changes occurring in human chromosomes during R-banding by Ba(OH)2 treatment were followed with the aid of bright-field and Nomarski interference contrast microscopy. It was found that the hot Ba(OH)2 pretreatment alone, i.e., without staining, caused a pattern of transverse ridges in the chromosomes that clearly corresponded to positive R-band regions. No chromosomal collapse could be seen during any stage of the R-banding procedure. Thus these events contrast with those observed in G-band formation with trypsin, where complete chromosomal collapse occurs after pretreatment and where staining is necessary to induce G-band ridges. The possible mechanism of R-band induction by Ba(OH)2 is discussed. It is proposed that the R-band ridges arise as a result of chromatin loss from the interband regions during the hot alkaline pretreatment.

Azure Stains↗