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Results for “Microscopy, Interference”

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At least 271 records · Page 15Linked to original sources

PubMed1999

On-stage selection of single round spermatids using a vital, mitochondrion-specific fluorescent probe MitoTracker(TM) and high resolution differential interference contrast microscopy.

The selection of individual round spermatids for round spermatid injection (ROSI), a prerequisite for the successful application of this infertility treatment, has been hampered by the ambiguous definition of a round spermatid and the lack of specific vital and non-vital markers. Using cells from rhesus monkey and bull, we describe a non-invasive method for the on-stage selection of individual round spermatids for ROSI, based on the polarized patterns of mitochondria, visualized in live round spermatid cells by epifluorescence microscopy after incubation with MitoTracker(TM), a vital, mitochondrion-specific fluorescent probe. The correct identification of live round spermatid was confirmed by the presence of the acrosomal granule or acrosomal cap in parallel observations by Nomarski differential interference contrast microscopy. The existence of mitochondrial polarization was first established by the labelling of MitoTracker-tagged round spermatids with spermatid-specific antibodies against proteins of nascent sperm accessory structures combined with antibodies against a nuclear pore complex component, known to disappear at the round spermatid stage. Using an inverted microscope equipped with epifluorescence, the round spermatids can be individually selected from a heterogeneous population of testicular cells labelled with MitoTracker dyes. A major advantage of this approach is that the dyes are incorporated into the paternal mitochondria, destined for rapid elimination after fertilization. In addition, the relatively high excitation and emission wavelengths of MitoTracker dyes are less harmful to DNA after their photon excitation. Before the appropriate clinical testing is conducted, the MitoTracker-based round spermatid selection may be instrumental in the training of clinical staff.

Animals↗

PubMed2004

Quantitative phase amplitude microscopy IV: imaging thick specimens.

The ability to image phase distributions with high spatial resolution is a key capability of microscopy systems. Consequently, the development and use of phase microscopy has been an important aspect of microscopy research and development. Most phase microscopy is based on a form of interference. Some phase imaging techniques, such as differential interference microscopy or phase microscopy, have a low coherence requirement, which enables high-resolution imaging but in effect prevents the acquisition of quantitative phase information. These techniques are therefore used mainly for phase visualization. On the other hand, interference microscopy and holography are able to yield quantitative phase measurements but cannot offer the highest resolution. A new approach to phase microscopy, quantitative phase-amplitude microscopy (QPAM) has recently been proposed that relies on observing the manner in which intensity images change with small defocuses and using these intensity changes to recover the phase. The method is easily understood when an object is thin, meaning its thickness is much less than the depth of field of the imaging system. However, in practice, objects will not often be thin, leading to the question of what precisely is being measured when QPAM is applied to a thick object. The optical transfer function formalism previously developed uses three-dimensional (3D) optical transfer functions under the Born approximation. In this paper we use the 3D optical transfer function approach of Streibl not for the analysis of 3D imaging methods, such as tomography, but rather for the problem of analysing 2D phase images of thick objects. We go on to test the theoretical predictions experimentally. The two are found to be in excellent agreement and we show that the 3D imaging properties of QPAM can be reliably predicted using the optical transfer function formalism.

Animals↗

PubMed1995

Motility of vinculin-deficient F9 embryonic carcinoma cells analyzed by video, laser confocal, and reflection interference contrast microscopy.

We have studied the motility of wild-type F9 and vinculin-deficient (5.51) mouse embryonal carcinoma cells. F9 cells extended filopodia at a rate of 61 ( +/- 18) nm/s over a distance of 3.18 (+/- 0.29) microns. In contrast, 5.51 cells exhibited filopodia which extended at a similar speed of 57 (+/- 17) nm/s but over a longer distance of 5.10 (+/- 2.14) microns. Cell-substratum contact areas of both cell types were examined by reflection interference contrast microscopy. Wild-type F9 cells had distinct close contacts (dark gray areas) at the cell periphery, whereas 5.51 cells had only a few light gray pinpoint contacts with the substrate. Confocal microscopy showed alpha-actinin to be localized along actin stress fibers in wild-type cells, and in 5.51 cells stress fibers were absent and alpha-actinin was associated with F-actin in the filopodia. beta 1-integrin, talin, and paxillin were concentrated in focal contacts in wild-type cells, but in 5.51 cells beta 1-integrin and talin were in patches under the plasma membrane and paxillin was diffusely distributed in the cytoplasm. We conclude that changes in cell shape and motility of 5.51 compared to wild-type F9 cells are due to the absence of vinculin even though there may be functions of other focal adhesion complex proteins, e.g., talin, linking the actin cytoskeleton to the plasma membrane.

Actinin↗

PubMed2000

Multiple Beam Interference Confocal Microscopy: Tool for Morphological Investigation of a Living Spermatozoon.

The interference pattern obtained from a multiple internal reflection of a spermatozoon, sandwiched between the glass plate and the cover plate, was focused on the objective of a scanning confocal microscope. According to optical path differences, morphological details were revealed. The combined features, namely improved resolution in z axis, originating from the interference pattern and the optical sectioning of the confocal scanning system, enhanced the resolution and contrast in an impressive manner. These features permitted unprecedented images of the spermatozoon to be obtained at 0.1 µm optical sectioning and reconstruction of three-dimensional (3-D) images. With the help of optical sectioning, it was possible to estimate the thickness and the morphological details of the spermatozoon.

Journal Article↗

PubMed1976

Morphology, ultrastructure, and mode of division of Mycoplasma fermentans, Mycoplasma hominis, Mycoplasma orale, and Mycoplasma salivarium.

The morphology of viable Mycoplasma fermentans, Mycoplasma hominis, Mycoplasma orale types 1 and 2, and Mycoplasma salivarium was studied in broth cultures by interference microscopy and in thin sections by electron microscopy. Only spherical cells were seen by interference microscopy. M. hominis had a capsule-like outer layer. Except for M. orale type 1, mycoplasmas in thin sections were 0.3-1 mum in diameter, with a bounding trilaminar membrane 7.5-10 nm thick. The mycoplasmas contained DNA fibrils and randomly distributed ribosomes. No polyribosomes were seen. Dividing mycoplasmas elongated slightly; the membrane invaginated, forming one bud. Sometimes M. hominis and M. salivarium produced one bud by elongation, and the bud was attached by a tube. This method of division is not considered as characteristic but rather as due to centrifugal force separating unfixed cells during preparation for electron microscopy. Cross-septa were never observed. In thin sections M. orale type 1 was elongated and without buds, an observation which suggested that preparation for electron microscopy distorted the mycoplasmas.

Cell Division↗

PubMed1991

Evaluating acrosome reaction steps with brightfield and differential interference contrast microscopy techniques.

Acrosomal integrity of bovine spermatozoa was evaluated using naphthol yellow S-erythrosine B stain with bright-field microscopy, .2% glutaraldehyde fixation with Nomarski optics, and live wet smears with Nomarski optics to compare the evaluation techniques. Four ejaculates per two Holstein bulls were enriched through a Percoll gradient, capacitated with heparin, and acrosome-reacted with lysophosphatidylcholine to prepare spermatozoa for in vitro fertilization. Spermatozoa samples were taken after each preparatory step, and percentage of intact acrosomes was evaluated with the stain, .2% glutaraldehyde, and live wet smear. Acrosomal integrity decreased with each preparatory step across all techniques. The decrease in integrity from heparin to lysophosphatidylcholine indicated that all methods detected the acrosome reaction. Technique means across preparation steps showed no differences. Correlations between microscopy techniques were high. The live wet smear used no fixation methods. Both the naphthol yellow S-erythrosine B stain and the .2% glutaraldehyde techniques employed fixation steps that may have introduced artifacts that could influence the data. The live wet smear evaluated by Nomarski optics allowed a comparable evaluation of the acrosome-reacted bovine spermatozoa. Comparing brightfield with differential interference contrast microscopy for detecting the acrosome reaction in bovine spermatozoa showed that both methods were equally accurate.

Acrosome↗

PubMed2004

Structure-function relationship of biological gels revealed by multiple-particle tracking and differential interference contrast microscopy: the case of human lamin networks.

Lamin B1 filaments organize into a thin dense meshwork underlying the nucleoplasmic side of the nuclear envelope. Recent experiments in vivo suggest that lamin B1 plays a key structural role in the nuclear envelope, but the intrinsic mechanical properties of lamin B1 networks remain unknown. To assess the potential mechanical contribution of lamin B1 in maintaining the integrity and providing structural support to the nucleus, we measured the micromechanical properties and examined the ultrastructural distribution of lamin B1 networks in vitro using particle tracking methods and differential interference contrast (DIC) microscopy. We exploit various surface chemistries of the probe microspheres (carboxylated, polyethylene glycol-coated, and amine-modified) to differentiate lamin-rich from lamin-poor regions and to rigorously extract local viscoelastic moduli from the mean-squared displacements of noninteracting particles. Our results show that human lamin B1 can, even in the absence of auxiliary proteins, form stiff and yet extremely porous networks that are well suited to provide structural strength to the nuclear lamina. Combining DIC microscopy and particle tracking allows us to relate directly the local organization of a material to its local mechanical properties, a general methodology that can be extended to living cells.

Diffusion↗

PubMed1998

Determination of Giardia cyst viability in environmental and faecal samples by immunofluorescence, fluorogenic dye staining and differential interference contrast microscopy.

Amongst the techniques suggested for the determination of Giardia cyst viability, the use of the fluorogenic dyes, fluorescein diacetate (FDA) and propidium iodide (PI) is the most often recommended, even though it appears to overestimate the number of viable cysts. In the present study, the replacement of FDA with 4',6-diamidino-2-phenylindole (DAPI) allowed simultaneous direct immunofluorescence with monoclonal antibody labelled with fluorescein isothiocyanate (MAb-FITC). Under these conditions, it was possible both to quantify the cysts according to the immunofluorescence technique, and to appreciate their viability by using fluorogenic dye staining (DAPI and PI) and differential interference contrast (DIC) microscopy. This method proved to be significantly better than the counting methods normally suggested. The technique has been applied to Giardia cysts recovered from faeces and wastewater sludge.

Animals↗

PubMed1982

Interaction between intracellular vacuoles and the cell surface analysed by finite aperture theory interference reflection microscopy.

Using finite aperture theory we have shown that localized very dark areas in the interference reflection images of Dictyostelium discoideum amoebae are due to the close intracellular approach of vesicles and tubular elements of the contractile vacuole system to the plasma membrane adjacent to the substratum. Vesicles interacting in this way become locally deformed to the planar contour of the substratum and are separated from the cell surface membrane by a constant approximately less than 0.1 micron of cytoplasm. Lamellar processes formed by these cells on very adhesive surfaces have identical dimensions. This minimal thickness may be a mechanical consequence of a contractile mechanism which pulls membranes together.

Animals↗

PubMed1990

The structure and dynamics of patch-clamped membranes: a study using differential interference contrast light microscopy.

We have developed techniques for micromanipulation under high power video microscopy. We have used these to study the structure and motion of patch-clamped membranes when driven by pressure steps. Patch-clamped membranes do not consist of just a membrane, but rather a plug of membrane-covered cytoplasm. There are organelles and vesicles within the cytoplasm in the pipette tip of both cell-attached and excised patches. The cytoplasm is capable of active contraction normal to the plane of the membrane. With suction applied before seal formation, vesicles may be swept from the cell surface by shear stress generated from the flow of saline over the cell surface. In this case, patch recordings are made from membrane that was not originally present under the tip. The vesicles may break, or fuse and break, to form the gigasealed patch. Patch membranes adhere strongly to the wall of the pipette so that at zero transmural pressure the membranes tend to be normal to the wall. With transmural pressure gradients, the membranes generally become spherical; the radius of curvature decreasing with increasing pressure. Some patches have nonuniform curvature demonstrating that forces normal to the membrane may be significant. Membranes often do not respond quickly to changes in pipette pressure, probably because viscoelastic cytoplasm reduces the rate of flow through the tip of the pipette. Inside-out patches may be peeled from the walls of the pipette, and even everted (with positive pressure), without losing the seal. This suggests that the gigaseal is a distributed property of the membrane-glass interface.

Animals↗

PubMedDate not supplied

Trichodina ctenophorii n. sp., a novel symbiont of ctenophores of the northern coast of the Gulf of Mexico.

Peritrich ciliates of the genus Trichodina are internal or external symbionts of invertebrate and vertebrate hosts. We describe here Trichodina ctenophorii n. sp., a symbiont of Mnemiopsis mccraydii and Beroë ovata (Phylum Ctenophora). The morphology of fixed and living specimens is revealed by silver impregnation, scanning electron microscopy, and differential interference microscopy. Distinguishing features of Trichodina ctenophorii include a denticular morphology composed of falcate, blunt-tipped blades, and long, straight thorns, with five pins per denticle. Trichodina ctenophorii is found only on the comb plates of these ctenophores. To the best of our knowledge, this is the first report of a trichodinid from the Gulf of Mexico and the first associated with ctenophores.

Alabama↗

PubMed1999

A new study of bacterial motion: superconducting quantum interference device microscopy of magnetotactic bacteria.

The recently developed "microscope" based on a high-Tc dc SQUID (superconducting quantum interference device) is used to detect the magnetic fields produced by the motion of magnetotactic bacteria, which have permanent dipole moments. The bacteria, in growth medium at room temperature, can be brought to within 15 micron of a SQUID at liquid nitrogen temperature. Measurements are performed on both motile and nonmotile bacteria. In the nonmotile case, we obtain the power spectrum of the magnetic field noise produced by the rotational Brownian motion of the ensemble of bacteria. Furthermore, we measure the time-dependent field produced by the ensemble in response to an applied uniform magnetic field. In the motile case, we obtain the magnetic field power spectra produced by the swimming bacteria. Combined, these measurements determine the average rotational drag coefficient, magnetic moment, and the frequency and amplitude of the vibrational and rotational modes of the bacteria in a unified set of measurements. In addition, the microscope can easily resolve the motion of a single bacterium. This technique can be extended to any cell to which a magnetic tag can be attached.

Biophysical Phenomena↗

PubMed2006

Expression of keratin 18 in the periderm cells of the lingual epithelium of fetal rats: visualization by fluorescence immunohistochemistry and differential interference contrast microscopy.

We examined the expression of keratin 18 (K18), by immunofluorescence staining, while monitoring morphological changes in the periderm on the lingual epithelium of rats by laser-scanning microscopy of epoxy resin-embedded, semi-ultrathin sections. We also examined differential interference contrast (DIC) images of the same sections to define the histology and morphology of the cells. It is difficult to visualize histological details of the fetal lingual epithelium of the rat on semi-ultrathin sections by light microscopy after immunohistochemical staining, because the histological structures in such sections cannot be distinguished by standard counterstaining. To solve this problem and to visualize keratin 18 (K18), we used a combination of immunofluorescence staining of semi-ultrathin sections and corresponding differential contrast (DIC) images, obtained by laser-scanning microscopy.

Animals↗

PubMed1994

On measuring the third dimension of cultured endothelial cells in shear flow.

The stress in the endothelial cells induced by blood flow depends on the waviness of the blood-endothelium interface and the slopes at the junctions of neighboring cells in the direction of flow. The height and slope in the third dimension of the living endothelial cells cannot be measured by ordinary optical and electron microscopy. Here we show that interference microscopy meets the challenge. We measured the geometry of cultured confluent human vascular endothelial cells in a flow, and we found that in a normal section parallel to the flow, the absolute values of the surface slopes at the cell junctions were 0.70 +/- 0.02 (SE) and 0.80 +/- 0.02 (SE) at the leading and trailing edges of the cells, respectively, in a culture medium of osmolarity 310 mosM with a shear stress of approximately 1 N/m2. A reversal of the flow direction led to a reversal of the slope pattern. An increase in medium osmolarity above 310 mosM induced an initial decrease in the slopes followed by a return to normal, whereas a decrease in the osmolarity had a reversed effect. These results, in light of our previous theoretical analyses, show that tensile stress exists in the endothelial cell membrane, and that the mechanism of tension accumulation is a reality. The accumulation is not 100% because the membranes are not smooth at the cell junctions.

Blood Viscosity↗

PubMedDate not supplied

Local cell membrane deformations due to receptor-ligand bonding as seen by reflection microscopy.

Understanding surface receptor clustering and redistribution processes at the cell-matrix contact zone requires detailed knowledge of the spatial integration of these molecules in the architecture of this complex interface. Here we present and discuss critically a procedure to extract such information combining reflection contrast microscopy (RCM) and reflection interference microscopy (RIM). As model system, we used living human umbilical vein endothelial cells (HUVEC) adhering to laminin-coated surfaces and investigated the distribution of the alpha2beta1 (CD29/CD49b) integrin at the contact zone of these cells. First, we applied freeze-fracture electron microscopy to gain information on microscopic details of the alpha2beta1 distribution at the contact zone. Next, we visualized and analyzed the overall lateral distribution of the integrins applying RCM using immunogold-labeling with 10 nm labels and a special silver enhancement technique. We found that RCM can be used to determine the lateral position of the marked receptor molecules to an accuracy of about 100-200 nm, instead of large morphological changes at the contact zone during silver enhancement. Finally, we combined RCM with RIM and analyzed the interference pattern of the contact zone around the label positions. Thus, we were able to detect changes of the average shape of the cell membrane due to receptor-ligand bonding of a size down to the resolution of the techniques.

Cell Adhesion↗

PubMed1997

Image processing for combined bright-field and reflection interference contrast video microscopy.

Image processing algorithms for automatic extraction of cell body contours and cell-substratum contacts from video images, which were obtained by bright-field microscopy and reflection interference contrast microscopy, respectively, are described. Double-view imaging, which combines these two optical techniques, is used to investigate the relationship between cell-to-substratum adhesion and cell shape changes during locomotion of the amoeboid cells of Dictyostelium discoideum. Contact areas of cells are extracted from reflection interference contrast images via a routine which performs binarisation on the basis of threshold estimation, as it is calculated by a histogram minimum method. Boundaries of cells are extracted from bright-field images by utilising and algorithm that includes background subtraction and binarisation based on texture discrimination by means of a rank operator.

Animals↗

PubMed1982

Light and electron microscopy of liver in hyperlipoproteinemic patients under long-term gemfibrozil treatment.

The effects of long-term gemfibrozil (Lopid) therapy on human liver structure are not known. Studies of this nature are becoming essential in determining the risk/benefit ratio since gemfibrozil is an effective agent for the control of hyperlipoproteinemia types IIa, IIb, and IV. Particularly, gemfibrozil is effective when dietary management or available therapeutic control fail to reduce serum cholesterol and triglycerides as well as normalizing the lipoprotein pattern. Percutaneous liver biopsies of 9 patients on long-term gemfibrozil therapy were evaluated by light microscopy, interference contrast optics and transmission electron microscopy. The distribution of patients according to lipoprotein phenotype was 3 Type IIa, 3 Type IIb, and 3 Type IV. Their lipoprotein patterns approached normal and the serum lipids were controlled during gemfibrozil therapy. By light microscopy, the lobular architecture and other parameters were within normal limits. Varying degrees of fatty change were found as would be expected. No preferential lobular disposition of the fat globules was evident. Coalescence of fat droplets, nuclear displacement and fatty cysts were noted. Differential interference contrast microscopy revealed several degrees of contrast amplitude in these droplets suggesting a heterogeneous lipid deposition in hepatocytes. The subcellular analysis revealed a moderate degree of glycogen deposition, absence of nuclear abnormalities and unremarkable mitochondria; the rough endoplasmic reticulum was not significantly altered and smooth surfaced membranes appeared proliferated. Detailed analysis of the peroxisome population showed matrix rarefaction, marginal plate formation and spurious densities though no significant proliferation occurred. Distribution of peroxisomes in hepatocytes varied widely from cell to cell and in different lobular areas. This study confirmed the association of hepatic fatty change with hyperlipoproteinemia irrespective of the pattern observed in circulating lipoproteins. Peroxisome proliferation, as seen in rodents when receiving gemfibrozil, did not occur and the structure of these subcellular organelles was not compromised. It was concluded that the long-term administration of this compound did not show adverse effects on the hepatocyte in hyperlipoproteinemia.

Gemfibrozil↗

PubMed1990

The microbial flora from root canals and periodontal pockets of non-vital teeth associated with advanced periodontitis.

Microflora from root canals and periodontal pockets of periodontally affected teeth were compared in order to elucidate the as yet unknown relationship between pulpal and periodontal disease. Caries-free teeth affected with advanced periodontitis and diagnosed as clinically dead by electric pulp testing were selected. The root canals and periodontal pockets were sampled, and the bacterial flora examined by both culture and interference microscopy. The results indicated that the aerobe/anaerobe ratio in the periodontal pocket was 0.23, while it was 0.0022 in the root canal, the large predominance of obligate anaerobes reflecting the anaerobic environment found in the root canal. Morphological classification obtained from interference microscopy showed similar proportions of morphotypes in the two sites. Results of anaerobic culture demonstrated a significantly higher rate of detection of facultative Streptococcus bacteria in the periodontal pocket than in the root canal. The predominant bacterial species common to both regions were Streptococcus, Peptostreptococcus, Eubacterium, Bacteroides, and Fusobacterium for obligate anaerobes. As for facultative anaerobes, Actinomyces and Streptococcus were detected predominantly in the periodontal pocket. The occurrence of micro-organisms common to both sites in this study suggests that the periodontal pocket may be a possible source of root canal infections.

Actinomyces↗