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At least 973 records · Page 54Linked to original sources
PubMed1998

Application of total internal reflection fluorescence microscopy to study cell adhesion to biomaterials.

Cell adhesion and function depend upon the formation of adhesive contacts between the cell and substrate. Determination of the cell substrate contact area is necessary in order to understand how biomaterial properties influence cell adhesion. In this review we describe the development and application of total internal reflection fluorescence microscopy (TIRFM) to quantify the separation distance of cells from a biomaterial surface. An approximate theory is presented for the straightforward calculation of separation distances when a fluor is placed in the cell membrane. The validity of this approach is discussed. TIRFM is compared to interference reflection microscopy and related techniques that measure cell/substrate separation distances. This approach is then applied to a number of important problems in cell substrate interactions, including changes in contact area and adhesion strength on biomaterial surfaces, analysis of bond strength, and real-time measurement of cell/substrate separation distances following exposure to flow.

Animals↗
PubMed1996

Kinesin movement on glutaraldehyde-fixed microtubules.

Glutaraldehyde-cross-linked microtubules were investigated as substrates for kinesin motility. Microtubules, formed in vitro from chicken brain tubulin, were stabilized with Taxol and chemically fixed with glutaraldehyde. The degree of tubulin monomer cross-linking as a function of time and glutaraldehyde concentration was characterized using polyacrylamide gel electrophoresis. Atomic force microscopy of fixed microtubules indicated that the cross-linking is sufficient to stabilize the gross structure of the microtubules against air drying or a distilled water challenge. Kinesin movement on immobilized, fixed microtubules was determined using a kinesin-coated bead motility assay observed with differential interference contrast microscopy. Within measurement error, kinesin bead movement velocities were independent of the degree of microtubule cross-linking. Binding affinity, however, decreased with increased cross-linking. Although air- and water-challenged microtubules did not support kinesin motility, a dilute suspension of glutaraldehyde-fixed microtubules in buffer supported kinesin motility for at least 2 days without any substantial degradation of activity. Fixed microtubules may be useful for several applications, including affinity purification of microtubule-associated proteins and motility measurements under extreme conditions of temperature and other variables.

Animals↗
PubMed1992

Interference image analysis of heat-shocked HeLa cells.

HeLa cells were studied with the interference microscope 1 h after heat shock at temperatures of 40 degrees C and 43 degrees C and also under conditions of recovery from the shock. The aim was to investigate changes in patterns of cellular dry mass distribution with the heat shock, based on variation of interference colours in interphase cells. A change in concentration and distribution of a partly high-salt-resistant material in the nuclear and perinuclear regions of the cells was found to be induced by the heat shock at 43 degrees C, a situation which reverted to control under recovery conditions. A similar interference image response was obtained for the heat shock assay at 40 degrees C, but it was detected only during the 4 h recovery period, suggesting that it could have been elicited later. The material induced by the heat shock and visualized by the analysis of interference images is assumed to be a part of the nuclear matrix-intermediate filament cell fraction.

Cell Nucleolus↗
PubMed1984

Contrast enhancement of the brownish horseradish peroxidase-activated 3,3'-diaminobenzidine tetrahydrochloride reaction product in black and white photomicrography by the use of interference filters.

An interference filter combination for contrast enhancement of the brownish, peroxidase-activated, 3,3'-diaminobenzidine tetrahydrochloride reaction product has been developed. This filter combination allows transmission of light in two narrow bands, representing those of the primary colors green (480-500 nm) and red (575-585 nm) as well as a broader band between 385-430 nm. The visual contrast enhancement of horseradish peroxidase-containing neurons caused by the interference filters was excellent. In particular, intracellular details were prominent in the presence of the interference filters compared with different blue absorption filters. The contrast enhancement was evaluated by microdensitometry of an Agfapan 25 emulsion onto which peroxidase-containing neurons in noncounterstained sections were exposed. The microdensitometry showed a neuron to background contrast enhancement of 30% compared to neutral gray filters and of 11% compared to a Kodak Wratten 80A blue absorption filter. The interference filters are available from the Laboratory of Technical Optics, the Technical University of Copenhagen, Building 307, Lundtoftevej 100, DK-2800 Lyngby, Denmark (order: DAB-interference filters, 30 mm). The interference filter principle can be used for contrast enhancement of other compound colors.

3,3'-Diaminobenzidine↗
PubMed1989

Dynamics of the endoplasmic reticulum and other membranous organelles in growth cones of cultured neurons.

The fluorescent lipophilic dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] was used to examine the distribution of membrane-bound organelles in growth cones of cultured rat sympathetic neurons. Within chemically fixed growth cones, intense DiOC6(3) fluorescence was localized predominately to the base or central region of growth cones. However, in most growth cones several thin DiOC6(3)-fluorescent processes radiated from the base into the periphery, and double fluorescence imaging of single growth cones indicated that these processes were highly colocalized (approximately 79%) with microtubules. The distribution of DiOC6(3) fluorescence in living growth cones was examined using low light-level fluorescence video microscopy. We observed thin fluorescent processes within the periphery of growth cones to undergo length excursions (extension/retraction) and to change orientation (move laterally). During growth cone advance, processes became progressively thicker and were gradually engulfed by the advancing fluorescent mass. When growth cones were viewed with video-enhanced differential interference contrast microscopy, the position of the fluorescent processes correlated with thickened extensions of central-type cytoplasm through which vesiclelike organelle transport often occurred. These observations indicate several features concerning the organization and movement of membranous organelles (MOs) in growth cones: (1) MOs are highly compartmentalized, the majority being localized to the growth cone base; (2) MOs advance into the periphery along distinct pathways probably associated with microtubules; (3) one or more thin continuous MOs, which most likely represent a thin tubular component of the endoplasmic reticulum, generally precedes advance of vesiclelike MOs along individual transport pathways; and (4) transport pathways with their associated MOs are spatially and temporally dynamic.

Animals↗
PubMed2005

Long-working-distance incoherent-light interference microscope.

We describe the design and operation of a long-working-distance, incoherent light interference microscope that has been developed to address the growing demand for new microsystem characterization tools. The design of the new microscope is similar to that of a Linnik interference microscope and thus preserves the full working distance of the long-working-distance objectives utilized. However, in contrast to a traditional Linnik microscope, the new microscope does not rely on the use of matched objectives in the sample and the reference arms of the interferometer. An adjustable optical configuration has been devised that allows the total optical path length, wavefront curvature, and dispersion of the reference arm to be matched to the sample arm of the interferometer. The reference arm configuration can be adjusted to provide matching for 5x, 10x, and 20x long-working-distance objectives in the sample arm. In addition to retaining the full working distance of the sample arm objectives, the new design allows interference images to be acquired in situations in which intervening windows are necessary, such as occur with packaged microsystems, microfluidic devices, and cryogenic, vacuum, or environmental chamber studies of microsystem performance. The interference microscope is compatible with phase-shifting interferometry, vertical scanning interferometry, and stroboscopic measurement of dynamic processes.

Equipment Design↗
PubMed2004

Image formation in low-coherence and confocal interference microscopes.

Image formation in the coherence probe microscope (CPM) and in optical coherence tomography (OCT) are compared. These systems differ in that CPM is a conventional interference microscope, but OCT is a confocal interference microscope. A major disadvantage of CPM for imaging through thick object structures is that there is no optical sectioning for the background image, which can saturate the detector. The behavior of the interference term in the presence of aberrations also exhibits some differences: Aberrations can be compensated in CPM, but not in OCT.

Equipment Design↗
PubMed1985

[Immunomorphological research on the changes in the focal contacts of cultured cells transformed by Kirsten sarcoma virus].

Alterations of focal contacts (FC) of NRK cultured cells transformed by Kirsten's sarcoma virus and by ts-mutant of this virus were examined immunomorphologically using a new antibody-exclusion method. In nontransformed NRK cells the FC are arranged in the form of a network near the cell edges; numerous single contacts are seen in the center. The FC are always present in both KNRK and tsKNRK cells at permissive temperature. However, their quantity is drastically reduced, especially in the center of the cells, as is often their size. It is suggested that expression of the product of viral oncogene interferes with maturation of the FC continuously formed at the cell edge.

Animals↗
PubMed2004

Direct observation of electrostatic microfields by four-electron-wave interference using two electron biprisms.

A new method has been developed for directly visualizing electromagnetic microfields in real time by the interference of four-electron waves obtained using a transmission electron microscope equipped with a field-emission gun and two electron biprisms. When one object wave and three reference waves interfere, equal-phase lines of the object wave are displayed as the intensity modulation of the interference fringes, in the manner same as in the three-electron-wave interference. The advantage of the four-electron-wave interference is that equal-phase lines are observed without precisely adjusting the angle between the two biprisms or rigorously controlling the fringe spacings of the two biprisms.

Holography↗
PubMed1984

Immunofluorescence localization of phosphotyrosine containing proteins in RSV-transformed mouse fibroblasts.

The localization of phosphotyrosine-containing proteins (P-TYR proteins) was studied by immunofluorescence microscopy employing affinity-purified azobenzyl phosphonate (ABP) antibodies, which specifically cross-react with phosphotyrosine. In Rous sarcoma virus (RSV)-transformed fibroblasts, after fixation followed by permeabilization with nonionic detergents, ABP antibodies gave a diffuse staining of the cytoplasm and specifically decorated restricted areas of the ventral plasma membrane corresponding to adhesion plaques, identified by interference reflection microscopy and staining with anti-vinculin serum. Specific decoration was also observed at the level of cell-cell contacts and at the tips of filopodial protrusions. Control non-transformed 3T3 mouse fibroblasts were not significantly stained by ABP antibodies. These findings show that, in RSV-transformed cells, proteins phosphorylated at tyrosine residues are found at cell-substratum and cell-cell contacts.

Animals↗
PubMed2005

Myosin Va transports dense core secretory vesicles in pancreatic MIN6 beta-cells.

The role of unconventional myosins in neuroendocrine cells is not fully understood, with involvement suggested in the movement of both secretory vesicles and mitochondria. Here, we demonstrate colocalization of myosin Va (MyoVa) with insulin in pancreatic beta-cells and show that MyoVa copurifies with insulin in density gradients and with the vesicle marker phogrin-enhanced green fluorescent protein upon fluorescence-activated sorting of vesicles. By contrast, MyoVa immunoreactivity was poorly colocalized with mitochondrial or other markers. Demonstrating an important role for MyoVa in the recruitment of secretory vesicles to the cell surface, a reduction of MyoVa protein levels achieved by RNA interference caused a significant decrease in glucose- or depolarization-stimulated insulin secretion. Similarly, expression of the dominant-negative-acting globular tail domain of MyoVa decreased by approximately 50% the number of vesicles docked at the plasma membrane and by 87% the number of depolarization-stimulated exocytotic events detected by total internal reflection fluorescence microscopy. We conclude that MyoVa-driven movements of vesicles along the cortical actin network are essential for the terminal stages of regulated exocytosis in beta-cells.

Animals↗
PubMed1993

Rous sarcoma virus-transformed cells develop peculiar adhesive structures along the cell periphery.

Alteration of the cell/substratum adhesive structures of rat fibroblasts (3Y1 cells) upon transformation by Rous sarcoma virus (RSV) was investigated by immunofluorescence microscopy. In serum-containing culture medium, 3Y1 cells developed focal adhesions as their main adhesive structures, while BY1 cells expressed peculiar close contacts along the cell periphery with the vitronectin receptor integrin, in addition to podosomes. These peripheral close contacts are referred to as the peripheral adhesions. The peripheral adhesions were observed as a darker region than podosomes by interference reflection microscopy. They were more easily destroyed by incubating the cells with RGD-containing peptide than were the focal adhesions. In contrast to focal adhesions and podosomes, actin bundles were not detected within the peripheral adhesions, where pp60v-src and tyrosine-phosphorylated proteins accumulated. Expression of the integrin was determined by the substratum composition when BY1 cells were cultured in serum-free culture medium. Under such conditions, BY1 cells expressed the peripheral adhesions within 3 hours on adhesion molecule-coated glass. On the other hand, in serum-containing medium, they first developed focal adhesions transiently at their early stage of adhesion, and then the peripheral adhesions were predominantly expressed within 12 hours. Podosomes were formed in a time course similar to that of the peripheral adhesions. These findings suggest that the peripheral adhesion is a class of stable adhesive structure distinct from the focal adhesion or podosome of BY1 cells. Similar close contact-type peripheral adhesions with the integrin were also observed in a variety of cultured cells such as normal fibroblasts at their logarithmic growth phase, phorbol ester-treated fibroblasts, and several malignant tumor cells, with poorly organized focal adhesions and stress fibers. These findings further suggest that the peripheral adhesions may be widely involved in the adhesion of cells that inadequately develop stress fibers and focal adhesions.

Actins↗
PubMed2001

Computational adaptive optics for live three-dimensional biological imaging.

Light microscopy of thick biological samples, such as tissues, is often limited by aberrations caused by refractive index variations within the sample itself. This problem is particularly severe for live imaging, a field of great current excitement due to the development of inherently fluorescent proteins. We describe a method of removing such aberrations computationally by mapping the refractive index of the sample using differential interference contrast microscopy, modeling the aberrations by ray tracing through this index map, and using space-variant deconvolution to remove aberrations. This approach will open possibilities to study weakly labeled molecules in difficult-to-image live specimens.

Animals↗
PubMed1977

A modified techinque to study leaf epidermis in Triticeae.

A rapid modified method is described to study stomata and other epidermal features without elaborate processing. The preparations are semipermanent and permit spatial relationship studies of both leaf surfaces from the same field. The cleared preparations are optically sectioned with brightfield or Nomarski interference contrast techniques without staining for contrast. The technique permits superior tissue resolution, clarity, minimal distortion of sections, and preservation of trichomes. The method is applicable to all species of Triticeae.

Histological Techniques↗
PubMed2003

Computer-synthesis of an interference color chart of human tear lipid layer, by a colorimetric approach.

PURPOSE: To synthesize an interference color chart for the specific tear lipid layer interference camera, DR-1, for the conversion of the tear film lipid layer thickness into color graphic information--that is, for the quantification of the tear interference image--by a colorimetric approach. METHODS: Because the color of the tear lipid layer interference image is visualized by a white light source interference phenomena, to produce gamma-corrected red, green, and blue (RGB) values of a specific interference color at a certain tear lipid film thickness, XYZ tristimulus values of the Commission Internationale de l'Eclairage (CIE) were obtained. XYZ tristimulus values were calculated from the light source spectrum of the DR-1 camera, the color-matching function of CIE, and the reflectance of the tear interference image in wavelengths ranging from 380 to 780 nm. These calculated interference colors were synthesized ranging from 0 nm to 1000 nm of lipid film thickness to produce a color chart. The applicability of the new color chart in the analysis of the lipid layer thickness was tested on a healthy control subject with normal tear function and a patient with dry eye who had aqueous tear deficiency and meibomian gland obstruction. RESULTS: The specific tear interference color chart for the DR-1 camera was obtained with RGB and XYZ tristimulus values. The interference chart ranged from 0 to approximately the 5th interference order. The interference colors from clinical DR-1 images could be converted to lipid thickness data by using the color chart system. CONCLUSIONS: A new tear interference color chart was developed in this study, which may be of benefit in converting tear interference color information to data describing the thickness of the tear film lipid layer.

Adult↗
PubMed1986

The structure of cytoplasm in directly frozen cultured cells. II. Cytoplasmic domains associated with organelle movements.

The relationship between organelle movement and cytoplasmic structure in cultured fibroblasts or epithelial cells was studied using video-enhanced differential interference contrast microscopy and electron microscopy of directly frozen whole mounts. Two functional cytoplasmic domains are characterized by these techniques. A central domain rich in microtubules is associated with directed as well as Brownian movements of organelles, while a surrounding domain rich in f-actin supports directed but often intermittent organelle movements more distally along small but distinct individual microtubule tracks. Differences in the organization of the cytoplasm near microtubules may explain why organelle movements are typically continuous in central regions but usually intermittent along the small tracks through the periphery. The central type of cytoplasm has a looser cytoskeletal meshwork than the peripheral cytoplasm which might, therefore, interfere less frequently with organelles moving along microtubules there.

Actins↗