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At least 343 records · Page 19Linked to original sources
PubMed1995

Determination of microtubule polarity in vitro by the use of video-enhanced differential-interference contrast light microscopy and Chlamydomonas flagellar axonemal pieces.

Microtubules nucleated by sea urchin sperm-tail axonemes have polar ends that differ both functionally and structurally but cannot be distinguished from one another when viewed by light microscopy. Ambiguity and circularity surround any classification of microtubule polarity by conventional methods. Chlamydomonas flagellar axonemal pieces have distinct morphological differences at their plus- and minus-ends, and microtubules nucleated from these pieces can be distinguished as plus- or minus-ended based on the morphological differences present in the Chlamydomonas flagellar axonemal pieces. Plus- and minus-ended microtubules were polymerized in this fashion and analyzed for differences in growth rates, shortening rates, and frequencies of transitions. The results were in good agreement with similar data generated by the more time-consuming and difficult use of kinesin-coated beads (R. J. Kowalski, and R. C. Williams, Jr. (1993) Cell Motil. Cytoskeleton 26, 282-290) to determine microtubule polarity. This is a relatively simple and effective method for determining the polarity of microtubules in vitro by video-enhanced differential-interference contrast light microscopy.

Animals↗
PubMed2005

Phase contrast and DIC illumination for AFM hybrids.

High-resolution optical microscopy is an essential pre-requisite for life science force microscopy, particularly for applications in cell biology and medicine. Identification and validation of cells is typically established with techniques like phase contrast microscopy or differential interference contrast microscopy. The option to select or monitor individual cells online with such light microscopy techniques while performing atomic force microscopy (AFM) measurements is therefore extremely beneficial. Here, we report two conceptually different strategies to implement these light microscopy techniques in a fully functional AFM head at the ultimate resolution of the Abbe diffraction limit.

Epithelial Cells↗
PubMed1986

Techniques for examining Pneumocystis carinii in fresh specimens.

Pneumocystis carinii was examined in fresh preparations of infected rat lung homogenates and tissue culture supernatants by a variety of light microscope techniques, vital dyes, and histologic stains. Phase-contrast microscopy, Nomarski interference-contrast microscopy, and bright-field microscopy with oblique illumination provided excellent views of P. carinii. Erythrosin B, and to a lesser extent trypan blue, were helpful in assessing organism viability. The use of Triton X-100-Giemsa stain permitted differentiation of the developmental stages in the P. carinii life cycle. The techniques developed here are easily adaptable to the microbiology laboratory and thus should have important clinical and research applications.

Animals↗
PubMed2006

Spiral interferogram analysis.

Interference microscopy using spatial Fourier filtering with a vortex phase element leads to interference fringes that are spirals rather than closed rings. Depressions and elevations in the optical thickness of the sample can be distinguished immediately by the sense of rotation of the spirals. This property allows an unambiguous reconstruction of the object's phase profile from one single interferogram. We investigate the theoretical background of "spiral interferometry" and suggest various demodulation techniques based on the processing of one single interferogram or multiple interferograms.

Journal Article↗
PubMed1991

Altered reactivity of hemolysate-treated cultured smooth-muscle cells from rabbit basilar artery determined by digital imaging microscopy.

During culture, smooth-muscle cells obtained from rabbit basilar arteries were examined for contractile activity by means of differential interference microscopy with a video analysis system (digital imaging microscopy system). This system proved useful for observing the contraction and ultrastructural changes of the living cells. Hemolysate-treated cells showed augmented responses to 5-hydroxytryptamine and leukotriene C4, but not to KCl. This augmented response diminished gradually during the culture period. Both a phospholipase C blocking agent, 2-nitro-4-carboxyphenyl-n,n-diphenylcarbamate (NCDC), and a myosin light chain kinase blocking agent, 1-(5-chloronaphthalenesulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), suppressed this augmented response. Protein kinase C activity of the cells, as measured by Western blot analysis, did not increase during the period of culture with hemolysate. The results obtained suggest that hemolysate had the following effects on the cells: 1) acute but gradual contraction of the cells; 2) augmentation of cellular responses to vasoactive agents; and 3) progressive contraction and morphological alteration of the cells. Possible mechanisms by which hemolysate exerts these effects are discussed, taking into consideration the interrelationship between these effects.

Animals↗
PubMed1966

Holomicrography: transformation of image during reconstruction of a posteriori.

Holomicrographs recorded through a microscope contain a hologram of the interior of the microscope, including the objective. During reconstruction of the microscopic image, modification of the aperture of the reconstructed objective produces the same alteration in the reconstructed image that would have occurred in the original image, if the actual objective had received the same modification. By this means, a single event holographed by bright-field microscopy may later be examined in reconstruction by dark-field, phase-contrast,or interference microscopy.

Eukaryota↗
PubMed1974

Electron microscope and experimental investigations of the neurofilamentous network in Deiters' neurons. Relationship with the cell surface and nuclear pores.

The assembly of filamentous elements and their relations to the plasma membrane and to the nuclear pores have been studied in Deiters' neurons of rabbit brain. Electron microscopy of thin sections and of ectoplasm spread preparations have been integrated with physicochemical experiments and differential interference microscopy of freshly isolated cells. A neurofilamentous network extends as a continuous, three-dimensional, semilattice structure throughout the ectoplasm, the "plasma roads," and the perinuclear zone of the perikaryon. This space network consists of approximately 90-A wide neurofilaments arranged in fascicles which are interconnected by an exchange of neurofilaments. The neurofilaments consist of intercoiled approximately 20-A wide unit-filaments and are associated through cross-associating filaments with other neurofilaments of the fascicle and with microfilaments. The approximately 20-50-A wide microfilaments display intimate associations with the plasma membrane and with the nuclear pores. Electron microscopy of thin sections from glycerinated and heavy meromyosin-treated Deiters' neurons shows that actin-like filaments are present in the pre- and postsynaptic regions of synapses terminating on these neurons. It is proposed that the neurofilamentous space network serves a transducing function by linking plasma membrane activities with the genetic machinery of the neuron.

Actins↗
PubMed1984

Serological and biochemical identification of a plasma membrane antigen specific to Leydig cells.

Purified mouse Leydig cells have been prepared from interstitial cell suspensions using a Percoll gradient procedure. The isolated cells are 88-95% pure as determined by light microscopy. Staining for 3-beta-hydroxysteroid dehydrogenase indicates that over 85% of the Leydig cells recognized by differential interference microscopy are also positive for this enzyme. After separation, the Leydig cells are viable by dye exclusion assays and exhibit normal in situ morphology when examined at the ultrastructural level. Leydig cell suspensions have been used to raise a polyclonal antiserum in rabbits. This antiserum, prior to absorption, reacts in indirect immunofluorescent studies with the surfaces of isolated Leydig cells, with testicular germ cells, with spermatozoa and with somatic cells such as splenocytes. Following absorption with lymphocytes and spermatogenic cells, the antiserum binds only to Leydig cell plasma membranes. Quantitative measurements with 125I-protein A confirm the specific labeling of Leydig cells by the absorbed antiserum. Biochemical identification of the Leydig cell plasma membrane antigen has been accomplished by immunoblotting polyacrylamide gel nitrocellulose transfers. A single major band of Mr approximately 40,000 is detected on one-dimensional transfers; two weakly reactive spots of Mr 43,000 and 45,000 are detected using two-dimensional immunoblots. Blotting experiments conducted using concanavalin A have identified the major Leydig cell constituents reactive with this lectin. The Leydig cell plasma membrane antigen(s) does not bind concanavalin A.

3-Hydroxysteroid Dehydrogenases↗
PubMed1998

An integrated approach to the study of living cells by atomic force microscopy.

We describe a technique for studying living cells with the atomic force microscope (AFM) in tapping mode using a thermostated, controlled-environment culture system. We also describe the integration of the AFM with bright field, epifluorescence and surface interference microscopy, achieving the highest level of integration for the AFM thus far described. We succeeded in the continuous, longterm imaging of relatively flat but very fragile cytoplasmic regions of COS cells at a lateral resolution of about 70 nm and a vertical resolution of about 3 nm. In addition, we demonstrate the applicability of our technology for continuous force volume imaging of cultured vertebrate cells. The hybrid instrument we describe can be used to collect simultaneously a diverse variety of physical, chemical and morphological data on living vertebrate cells. The integration of light microscopy with AFM and steady-state culture methods for vertebrate cells represents a new approach for studies in cell biology and physiology.

Animals↗
PubMed1976

Red blood cell shapes as explained on the basis of curvature elasticity.

Assuming that the shape of red blood cells is controlled by the curvature elasticity of the surrounding membrane, we fit theoretical shapes to the contours Evans and co-workers determined by interference microscopy. Very good agreement is obtained for disc shapes. The fit is not so good for less common shapes, which may result from Evans' parametric representation and from the interference of shear elasticity.

Elasticity↗
PubMed1995

A transmission electron microscopic (TEM) method for determining structural colors reflected by lizard iridophores.

Iridescent tissue colors are thought to be produced by iridophores through the optical phenomenon of thin-layer interference. Land and others have shown that structural features, predominantly reflecting platelet width and the cytoplasmic spacing between layers of platelets, determine the wavelength of light maximally reflected by this mechanism in iridophores. Some researchers have used interference microscopy to estimate these structural parameters, but the most direct measurement technique should be transmission electron microscopy (TEM). Transmission electron microscopy (TEM) has associated processing artifacts (particularly cytoplasmic shrinkage) that preclude direct measurement of ultrastructure, but if a number of assumptions are made, reflected wavelengths can be predicted. A thin-layer interference model and its associated assumptions were tested using TEM measurements of iridophores from several brightly colored tissues of each of three lizards (Sceloporus jarrovi, S. undulatus erythrocheilus, and S. magister). In all the instances examined when the contribution of the pigments present were accounted for, tissue color corresponded with predicted iridophore reflectances from the model. Finally, if the model and its assumptions are assumed to be correct, the amount of iridophore cytoplasmic shrinkage as a result of TEM processing can be calculated.

Animals↗
PubMed1995

Selective stimulation of epithelial cells in colonic crypts: relation to active chloride secretion.

Stimulation of Cl secretion by prostaglandin E2 (PGE2) was measured as the short-circuit current (Isc) across isolated epithelium of the rabbit distal colon. Cellular morphology of columnar and goblet cells during secretion was monitored using light and electron microscopy. Stimulation by PGE2 altered epithelial cell morphology only by a reduction of vacuolar space in the apical pole of crypt columnar cells, consistent with release of vacuole contents. Imaging of isolated crypts using differential interference microscopy confirmed the release of material from columnar cells during the onset of secretion. Inhibition of Cl secretion with the loop diuretic bumetanide did not block vacuole release. The actin filament-disrupting agent, cytochalasin, reduced the PGE2-stimulated Isc by 40% and blocked emptying of the vacuolar space. These electrical and morphological results indicate that the process of active ion secretion is associated with release of the macromolecular contents from apical vacuoles through a mechanism involving the cytoskeleton. In addition, this relationship supports the concept that vacuolated columnar cells of the crypts of Lieberkühn are the cell type that secretes Cl in response to PGE2.

Animals↗
PubMed2002

Interferometry in flow to sort unstained X- and Y-chromosome-bearing bull spermatozoa.

BACKGROUND: It was found earlier that the difference in volume between unstained X- and Y-chromosome-bearing sperm heads could be detected using interference microscopy in visible light. This could be the basis for an alternative to the conventional method to sort X and Y sperm, which uses DNA staining and ultraviolet (UV)-excitation that may be harmful to sperm cells. A novel technique is introduced combining interferometry with flow cytometry. MATERIALS AND METHODS: Interference optics were built into an existing flow cytometer/cell sorter and used to sort fresh unstained bull sperm cells on the basis of their head volume. Sorted fractions were stained with a DNA stain, and reanalyzed using the conventional method. RESULTS: Purities between 60-66% were found in both X- and Y-enriched fractions. It was possible to sort up to 300 cells per second. The system was found to be less sensitive to the orientation of sperm cells than the conventional method. CONCLUSIONS: Interferometry can be combined with flow cytometry and used to obtain significantly enriched fractions of X- and Y-bearing sperm without staining and UV light. Sorting speeds and purities at this point, however, are much lower than with the conventional method.

Animals↗
PubMed1999

Determination of age from cemental incremental lines for forensic dentistry.

Determination of age from cemental incremental lines was evaluated in intact teeth obtained from 17 individuals aged 23-77 years. Mineralized 100 microm cross sections were subjected to one of three treatments: unstained, stained with Villanueva's blood stain, and stained with acridine orange. Ideal areas were selected by light microscopy and photographed. Countability of incremental lines from photographic enlargements were evaluated. The average number of years required for the eruption of a particular tooth was added to the incremental lines count to determine the estimated age for that individual. Results obtained from unstained mineralized 100 microm thick cross sections using differential interference microscopy (Nomarsky) provided the most countable lines. The accuracy and repeatability of the method is not dependent on tooth type or location, but on the average obtained from making as many counts as possible. This method can be applied to general populations regardless of systemic or periodontal health.

Acridine Orange↗
PubMed1985

Adhesiveness and distribution of vinculin and spectrin in retinal pigmented epithelial cells during growth and differentiation in vitro.

Colonies of chick retinal pigmented epithelial (RPE) cells offer an excellent model system for studying the organization of cytoskeleton in sheets of differentiating epithelial cells. The cells occupying the center of the colony resemble RPE cells in vivo and are cuboidal, pigmented, and relatively nonadherent while those toward the periphery gradually become flatter, nonpigmented, motile, and strongly adherent to the substratum. Immunofluorescence microscopy with antiserum against chicken erythrocyte alpha-spectrin reveals that this protein is present in the cortex of RPE cells in all parts of the colony. It is neither concentrated in, nor excluded from the regions occupied by the major microfilament bundles, and its distribution is not related to the adhesion patterns visualized by surface reflection interference microscopy. In contrast, the distribution of vinculin is closely correlated with the adhesiveness of RPE cells in different parts of the colony. Immunofluorescence microscopy reveals that in the RPE cells vinculin may be diffusely distributed in the cytoplasm; present in a cortical band outlining the cell borders; and present in focal contacts and adhesions. The distribution of vinculin is affected by the length of time the colonies grow in culture, by the degree of cell packing and by the adhesiveness of cells to the substratum. In RPE cells grown in vitro for short periods (less than or equal to 3 days) vinculin is found in focal contacts and adhesions in both the undifferentiated, well spread peripheral cells as well as in the differentiated, polygonally packed central cells of the colony. In RPE cells cultured for longer periods (greater than or equal to 14 days) vinculin is present in focal contacts and adhesions only in strongly adherent, undifferentiated cells at the edge of the colony. In packed central cells of both short- and long-term cultures vinculin is found in the cortical band which circumscribes the apical ends of cells at the level of the adherens type intercellular junctions. Its appearance in the cortical bands does not depend on the length of time the colonies are grown in vitro but on the presence of cell-cell contacts resulting from an increased degree of cell packing within the central part of the colony. These results are discussed in relation to the development and the role of extracellular matrix in determining the adhesiveness of RPE cells in vitro.

Animals↗
PubMed2006

Estimation of polymer-surface interfacial interaction strength by a contact AFM technique.

Atomic force microscopy (AFM) measurements were employed to assess polymer-surface interfacial interaction strength. The main feature of the measurement is the use of contact-mode AFM as a tool to scratch off the polymer monolayer adsorbed on the solid surface. Tapping-mode AFM was used to determine the depth of the scraped recess. Independent determination of the layer thickness obtained from optical phase interference microscopy (OPIM) confirmed the depth of the AFM scratch. The force required for the complete removal of the polymer layer with no apparent damage to the substrate surface was determined. Polypropylene (PP), low-density polyethylene (PE), and PP-grafted-maleic anhydride (PP-g-ma) were scraped off silane-treated glass slabs, and the strength of surface interaction of the polymer layer was determined. In all cases it was determined that the magnitude of surface interaction force is of the order of van der Waals (VDW) interactions. The interaction strength is influenced either by polymer ability to wet the surface (hydrophobic or hydrophilic interactions) or by hydrogen bonding between the polymer and the surface treatment.

Adsorption↗
PubMed2006

Chromosomes can congress to the metaphase plate before biorientation.

The stable propagation of genetic material during cell division depends on the congression of chromosomes to the spindle equator before the cell initiates anaphase. It is generally assumed that congression requires that chromosomes are connected to the opposite poles of the bipolar spindle ("bioriented"). In mammalian cells, we found that chromosomes can congress before becoming bioriented. By combining the use of reversible chemical inhibitors, live-cell light microscopy, and correlative electron microscopy, we found that monooriented chromosomes could glide toward the spindle equator alongside kinetochore fibers attached to other already bioriented chromosomes. This congression mechanism depended on the kinetochore-associated, plus end-directed microtubule motor CENP-E (kinesin-7).

Animals↗