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PubMed1979

Application of differential interference contrast with inverted microscopes to the in vitro perfused nephron.

The study of in vitro perfused individual nephron segments requires a microscope which provides: (1) easy access to the specimen for measurement of cellular solute flux and voltage; (2) an image with high resolution and contrast; (3) optical sectioning of the object at different levels; and (4) rapid recording of the morphological phenomena. This paper describes an example of commercially available apparatus meeting the above requirements, and illustrates its efficiency. The microscope is of the inverted type (Zeiss IM 35) equipped with differential-interference-contrast (DIC) with a long working distance, and an automatically controlled camera system. The microscopic image exhibits cellular and intercellular details in the unstained transporting mammalian nephron segments despite their tubular structure and great thickness and makes obvious function-structure correlations (e.g. cell volume changes); luminal and contraluminal cell borders are well resolved for controlled microelectrode impalement.

Animals↗
PubMed2006

Biomolecular interaction analysis under electrophoretic flow conditions.

Combining the advantages of electrophoresis with the advantages of biomolecular interaction analysis (BIA) enables the biospecific detection of separated molecules; for example it permits differentiation between a complementary single-stranded DNA and a single nucleotide polymorphism. In order to integrate these two techniques, it is necessary to investigate whether it is possible to detect a biomolecular interaction under electrophoretic flow conditions. To this end a novel detection system was developed for electrophoresis that utilizes a label-free and time-resolved detection technique: reflectometric interference spectroscopy (RIfS). The biological functions of important analytes were investigated using this system. Although RIfS can be used as a postcolumn detector, it is also possible to use it to detect relevant substances under electrophoretic flow conditions. DNA-LNA, biotin-streptavidin and protein-protein interactions were detected using this coupled electrophoresis-RIfS set-up.

Antibodies↗
PubMedDate not supplied

Cryptosporidium parvum life cycle in suckling mice: a Nomarski interference-contrast study of a human-derived strain.

Cryptosporidiosis has emerged as one of the life-threatening opportunistic enteric infections in HIV-infected persons. To date, Cryptosporidium parvum is known to infect man via person-to-person or zoonotic transmission. We studied the sequential stages of the life cycle of C. parvum by Normarski interference-contrast microscopy in fresh gut specimens of newborn mice, infected with a strain derived from an AIDS patient with cryptosporidial diarrheal enteritis. Many 4- to 5-day-old suckling BALB/C mice were orally inoculated with 1 x 10(6) oocysts, obtained by acid flocculation of the patient's stools. The animals were sacrificed from 4 to 96 h post-infection and the ileum was examined microscopically. All stages of the asexual life cycle of C. parvum, from excysted sporozoites in the intestinal lumen through the development of type II mature meronts, 12- to 72-h post-infection, were documented by extemporaneous microscopic evaluation of fresh gut samples. The sexual cycle, characterized by the appearance of micro- and macrogametocytes, followed by a zygote developing into a sporulated oocyst, was documented as early 48-h post-infection. Our Nomarski interference-contrast observations on the life cycle of C. parvum yielded data comparable with those originally published by Current and Reese, and confirm the results of previous electron microscopic studies performed by several other authors.

Acquired Immunodeficiency Syndrome↗
PubMed1981

Scanning electron microscopy, autolysis, and irradiation as techniques for studying small intestinal morphology.

Examination of autolysed control mouse small intestine using scanning electron microscopy has revealed details of the connective tissue components of the mucosa. The cores of the villi are seen collapsed across the intervillous basin. Crypts of Lieberkuhn are seen as tubular channels stretching down from the intervillous basin. Sometimes the crypts are split in two by a connective tissue septum. The mouths of the crypts of Lieberkuhn are, in general, arranged in double rows between the single rows of villi. The ratio of number of crypts to numbers of villi was calculated as 5.01:1. This is close to the figure of 4.53:1, as quoted by Smith & Jarvis (1980) who used differential interference contrast microscopy to investigate the crypt to villus ratio. After radiation, the severe drop in the number of crypt mouths can be clearly seen by the combination of autolysis and scanning electron microscopy: the rows of crypt mouths between the villi have been lost, and many crypt mouths have been occluded by stromal tissue. The arrangement of the crypt mouths and the observation of mucosal abnormalities after irradiation have led to the postulation that cells leaving the crypt mouths move in a spiral manner towards and then up the villous surface: this postulated movement might imply an asymmetry in some properties of enterocytes. The use of scanning electron microscopy in conjugation with autolysis and irradiation has thus forced a critical re-examination of the relationships between crypts and villi.

Animals↗
PubMed2002

The distribution of caveolin-3 immunofluorescence in skeletal muscle fibre membrane defined by dual channel confocal laser scanning microscopy, fast Fourier transform and image modelling.

Membrane domains rich in caveolin-3 overlie sarcomeric actin in skeletal muscle. The membrane exhibits a regular array of caveolin-3 immunofluorescence using confocal laser scanning microscopy (CLSM). Fourier analysis of tissue imaged by CLSM accurately defines a repeating intensity with a long-axis spacing of 1.48 microm confirmed by measurement of direct images. Reverse fast Fourier transform (FFT) and image-modelling allow reconstruction of the pattern. Mathematical modelling has allowed replication of several features of the FFT, including the second order maxima that confirm the relatively high information content of the original images. Measurements of membrane-pattern primary long-axis spacings are consistent with our measurements of the I-band sarcomere repeat in similarly prepared specimens labelled with fluorescent phalloidin or imaged using differential interference contrast microscopy. Dual-channel CLSM analysis of the sarcomeric banding pattern of actin and the repeating pattern of muscle fibre membrane caveolin showed that caveolae overlie the I-band. The anti-caveolin immunofluorescence is deficient over the Z-disc and maximal toward each of the I-band extremities. A mechanism of membrane shape change in which membrane-lipid molecules are interposed between more stable anchored rafts associated with caveolae can be envisaged. Thus, increasing girth and reducing length of the sarcolemma in rapid contraction may be explained.

Animals↗
PubMed1981

A transmembrane relationship between fibronectin and vinculin (130 kd protein): serum modulation in normal and transformed hamster fibroblasts.

Using electron microscopy, we had previously demonstrated a very close transmembrane relationship between actin microfilaments and fibronectin fibrils, termed the fibronexus. Since vinculin, a recently discovered intracellular protein, is localized at the membrane-insertion regions of actin fibers, we studied its possible relationship to fibronectin and the fibronexus. Using double-label immunofluorescence microscopy, we have observed that the distributions of vinculin and fibronectin are strikingly coincident in normal Nil 8 hamster fibroblasts arrested in the G1 phase of the cell cycle, and in HSV-transformed Nil hamster cells treated with purified fibronectin after culturing in 0.3% serum. Extensively spread Nil 8 cells have numerous vinculin-positive focal patches, which are localized either directly over or in tandem with fibronectin fibers at the ventral surface. However, fibronectin and vinculin do not exhibit this relationship in Nil 8 cells grown in 5% serum. These vinculin patches closely resemble the vinculin plaques that Geiger found to be dark under interference-reflection microscopy, suggesting that fibronectin is associated with substrate-adhesion plaques in arrested cells. Fibronectin treatment of the HSV-transformed Nil cells cultured in a low concentration of serum results in the formation of ventral microprocesses, exhibiting an extraordinary congruence of vinculin and fibronectin staining. In addition, these cells bind matrix-like arrangements of fibronectin on their dorsal surface at sites of cell-cell interaction that are vinculin-negative. These results imply that two distinct types of fibronexuses may exist: a ventral substrate-adhesive nexus consisting of fibronectin, vinculin and actin, and a dorsal association matrix fibers. Transmembrane vinculin-fibronectin associations are evidently sensitive to the growth state of the cell.

Animals↗
PubMed2004

Differential phase optical coherence probe for depth-resolved detection of photothermal response in tissue.

We describe a differential phase low-coherence interferometric probe for non-invasive, quantitative imaging of photothermal phenomena in biological materials. Our detection method utilizes principles of optical coherence tomography with differential phase measurement of interference fringe signals. A dual-channel optical low-coherence probe is used to analyse laser-induced thermoelastic and thermorefractive effects in tissue with micrometre axial resolution and nanometre sensitivity. We demonstrate an application of the technique using tissue phantoms and ex-vivo tissue specimens of rodent dorsal skin.

Acoustics↗
PubMedDate not supplied

Increased conspicuity of intraventricular lesions revealed by three-dimensional constructive interference in steady state sequences.

We describe our preliminary experience with the three-dimensional constructive interference in steady state (3D-CISS) sequence for the evaluation of intraventricular lesions. Cyst walls, extent and margins of tumors, and intratumoral cystic structures were clearly depicted on 3D-CISS images. The 3D-CISS sequence can offer additional information to conventional MR studies to define intraventricular lesions better.

Adult↗
PubMed1987

Ethanol treatment inhibits mesoderm cell spreading in the gastrulating chick embryo.

Gastrulating chick embryos in culture were treated with ethanol solutions, following which the mesoderm cells migrating from the primitive streak were examined by scanning electron microscopy. Morphometric analysis of cell shape showed that mesoderm cells from treated embryos were significantly more rounded and therefore less well spread than controls, and showed fewer filopodial contacts with the overlying basement membrane. This result was only obtainable for cells leading the migration from the primitive streak, since the following cells in the mesodermal mass apparently did not show this difference. The ethanol concentration required to obtain a reliable effect was 5%, while lower concentrations produced highly variable results. The mesoderm cells were also examined for their in vitro responsiveness to ethanol by investigating their adhesiveness and cytoskeleton. No effect was observed on cell-glass adhesion as judged by interference reflection microscopy using up to 1% ethanol. This concentration did, however, disrupt the actin cytoskeleton of cultured cells when stained with NBD-phallacidin, but lower concentrations were ineffective. It is concluded that ethanol treatment of cultured embryos has a significant effect on the substratum relationships of some migrating mesoderm cells.

Animals↗
PubMed1983

Distribution of filipin-sterol complexes on cultured muscle cells: cell-substratum contact areas associated with acetylcholine receptor clusters.

Specialized areas within broad, close, cell-substratum contacts seen with reflection interference contrast microscopy in cultures of Xenopus embryonic muscle cells were studied. These areas usually contained a distinct pattern of light and dark spots suggesting that the closeness of apposition between the membrane and the substratum was irregular. They coincided with areas containing acetylcholine receptor clusters identified by fluorescence labeled alpha-bungarotoxin. Freeze-fracture of the cells confirmed these observations. The membrane in these areas was highly convoluted and contained aggregates of large P-face intramembrane particles (probably representing acetylcholine receptors). If cells were fixed and then treated with the sterol-specific antibiotic filipin before fracturing, the pattern of filipin-sterol complex distribution closely followed the pattern of cell-substratum contact. Filipin-sterol complexes were in low density in the regions where the membrane contained clustered intramembrane particles. These membrane regions were away from the substratum (bright white areas in reflection interference contrast; depressions of the P-face in freeze-fracture). Filipin-sterol complexes were also in reduced density where the membrane was very close to the substratum (dark areas in reflection interference contrast; bulges of the P-face in freeze-fracture). These areas were not associated with clustered acetylcholine receptors (aggregated particles). This result suggests that filipin treatment causes little or no artefact in either acetylcholine receptor distribution or membrane topography of fixed cells and that the distribution of filipin-sterol complexes may closely parallel the microheterogeneity of membranes that exist in living cells.

Animals↗
PubMed1983

Thickness variations within individual paraffin and glycol methacrylate sections.

The object of this study was to investigate whether there are intra-section thickness variations in individual paraffin and glycol methacrylate (GMA) sections. Using steel or glass knives sections were cut from liver and urinary bladder. Section thickness variations were measured with an interference microscope and amounted to 1-3 microns within individual paraffin sections and 0.3 microns within GMA sections. The results were confirmed by observations on sections which had been re-embedded and re-sectioned. Some of the variations within the paraffin sections were associated with the cell nuclei. It is concluded that GMA sections are much smoother than paraffin sections and thus more suitable for quantitative histological studies.

Animals↗
PubMed1992

Neuronal injury evoked by depolarizing agents in rat cortical cultures.

Chemical depolarization is often used to study neurotransmitter release. Three commonly used depolarizing agents, veratridine, potassium, and glutamate, were evaluated for neurotoxicity. Neuronal survival and lactate dehydrogenase efflux were measured to assay irreversible injury. In addition, video-enhanced differential interference contrast microscopy was used to measure acute neuronal swelling. We found that lactate dehydrogenase efflux and cell death associated with exposure to potassium and glutamate could be blocked by the competitive N-methyl-D-aspartate antagonist amino-phosphonovaleric acid. Neuronal swelling was observed with all three agents, and could not be blocked by amino-phosphonovaleric acid. These results suggest multiple mechanisms of neuronal injury accompanying chemical depolarization. A 60-min exposure to 100 microM veratridine increased lactate dehydrogenase appearing in the medium at the end of this exposure to 615% of control and produced a 62% loss of neurons after 20-24 h. These effects could not be blocked by amino-phosphonovaleric acid at 500 microM. Differential interference contrast imaging revealed acute neuronal swelling in response to veratridine within 5 min of exposure, and this swelling could not be blocked by amino-phosphonovaleric acid. A 60-min exposure to medium supplemented with 50 mM KCl caused a lactate dehydrogenase efflux of 204% of control and produced a 48% loss of neurons. Amino-phosphonovaleric acid blocked both the neuronal loss and the excess lactate dehydrogenase efflux. In addition, differential interference contrast monitoring showed no KCl-evoked swelling. In contrast, isotonic substitution of 50 mM KCl for NaCl resulted in acute swelling which could not be blocked by amino-phosphonovaleric acid, in addition to neuronal death and lactate dehydrogenase release. Glutamate was, as expected, neurotoxic, and as has been shown before, this toxicity could be blocked by amino-phosphonovaleric acid. Observation of neurons exposed to 300 microM glutamate revealed that this treatment was invariably associated with neuronal swelling. In the presence of amino-phosphonovaleric acid, 81% of neurons swelled to greater than 110% by 30 min exposure to glutamate. These results suggest that experimental paradigms which investigate the effects of chemical depolarization upon central neurons are likely to be associated with reversible and irreversible forms of injury. This is of special importance to any study of the mechanisms of release of substances from central neurons.

Animals↗
PubMed1989

Cytoskeleton, adhesion, and extracellular matrix of fetal human retinal pigmented epithelial cells in culture.

Small sheets of human retinal pigmented epithelial (RPE) cells isolated from 6- to 9-week-old fetal human eyes were used to establish RPE cells in culture. After 4-5 days in culture the RPE cells in various parts of a colony were examined by immunofluorescence and fluorescence microscopy to determine the distribution of the cytoskeletal proteins tubulin, myosin, F-actin, vimentin, and cytokeratin and the extracellular matrix proteins laminin and type IV collagen. The extracellular matrix components laminin and type IV collagen, which are abundant underneath the RPE cells in the centre of the colony, progressively decrease in amount towards the edge of the colony. Microtubules labeled by antisera to tubulin were present in the cytoplasm of RPE cells throughout the colony. F-actin and myosin were found in stress fibres in the RPE cells at the edge of the colony and in compact circumferential rings circumscribing the apical ends in RPE cells in the centre of the colony. Examination of cell-substratum adhesion patterns by interference reflection microscopy showed that focal contacts were present only in RPE cells at the edge of the colony where they were located at the ends of the stress fibres. The large focal adhesions previously observed in colonies of chick RPE cells, however, were not seen in human RPE cells. Human RPE cells throughout the colony had cytokeratin-containing intermediate filaments as well as vimentin-containing intermediate filaments, whereas only the latter were observed in chick RPE cells. These results indicate that in the general organization of the cytoskeleton and in the distribution of the extracellular matrix components, laminin and type IV collagen, colonies of human RPE cells are similar but not identical to colonies of chick RPE cells.

Actin Cytoskeleton↗
PubMed1989

Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response.

Cell-substratum adhesion strengths have been quantified using fibroblasts and glioma cells binding to two extracellular matrix proteins, fibronectin and tenascin. A centrifugal force-based adhesion assay was used for the adhesive strength measurements, and the corresponding morphology of the adhesions was visualized by interference reflection microscopy. The initial adhesions as measured at 4 degrees C were on the order of 10(-5)dynes/cell and did not involve the cytoskeleton. Adhesion to fibronectin after 15 min at 37 degrees C were more than an order of magnitude stronger; the strengthening response required cytoskeletal involvement. By contrast to the marked strengthening of adhesion to FN, adhesion to TN was unchanged or weakened after 15 min at 37 degrees C. The absolute strength of adhesion achieved varied according to protein and cell type. When a mixed substratum of fibronectin and tenascin was tested, the presence of tenascin was found to reduce the level of the strengthening of cell adhesion normally observed at 37 degrees C on a substratum of fibronectin alone. Parallel analysis of corresponding interference reflection micrographs showed that differences in the area of cell surface within 10-15 nm of the substratum correlated closely with each of the changes in adhesion observed: after incubation for 15 min on fibronectin at 37 degrees C, glioma cells increased their surface area within close contact to the substrate by integral to 125-fold. Cells on tenascin did not increase their surface area of contact. The increased surface area of contact and the inhibitory activity of cytochalasin b suggest that the adhesive "strengthening" in the 15 min after initial binding brings additional adhesion molecules into the adhesive site and couples the actin cytoskeleton to the adhesion complex.

Animals↗
PubMed1976

Contrast enhancement of negatively stained macromolecules and biomembranes by single sideband phase contrast interference.

A straightforward procedure is described for the production of contrast enhancement of negatively stained macromolecules and biological membranes by single sideband phase contrast interference (electron optical shadowing). The instrumental adjustment required to produce this type of phase contrast illumination is readily achieved by beam deflection from the strioscopic (dark field) mode. Part of the hollow cone of electrons from the annular condenser aperture that are unscattered by the specimen are permitted to pass through the objective aperture and interfere with the scattered beam. The electron optical shadowing effect is produced because only one side of the unscattered beam is used. Careful adjustment of the beam tilt control, with the ability to tilt in any azimuth, allows optimal illumination conditions to be achieved. The results presented show the increased image contrast obtained using as specimens the purified cylindrical macromolecule from human erythrocyte membranes, purified nuclear envelopes and collagen fibres.

Animals↗
PubMed1977

[Quantitative study of the peripheral nerve in semi-thin section by the Nomarski interference contrast technic].

The Normarski interference-contrast microscope is well suited to observations of transverse thick sections of epon-embedded peripheral nerves. The quantitative study of myelinated fibers is possible on photomicrographs with a final magnification of X 500. The induced distorsion is very small as compared with the results obtained by conventional bright field and positive phase contrast optics. The comparative study of the same field with the three devices allows a better interpretation of marginal pictures.

Humans↗
PubMed2002

Electron interference: mystery and reality.

Interference of electron waves has developed from a fascinating phenomenon in basic physics to a key method for the highly sophisticated investigation of both electric and magnetic structures in solid-state materials. After more than 20 years of development, electron holography in the transmission electron microscope is now a very powerful technique for the analysis of micro-fields down to atomic dimensions. The applications extend from highly sensitive measurements in semiconductor technology to the quantitative characterization of atomic structures.

Electromagnetic Fields↗
PubMed2002

High-resolution full-field optical coherence tomography with a Linnik microscope.

We describe an original microscope for high-resolution optical coherence tomography applications. Our system is based on a Linnik interference microscope with high-numerical-aperture objectives. Lock-in detection of the interference signal is achieved in parallel on a CCD by use of a photoelastic birefringence modulator and full-field stroboscopic illumination with an infrared LED. Transverse cross-section (en-face, or XY) images can be obtained in real time with better than 1-microm axial (Z) resolution and 0.5-microm transverse (XY) resolution. A sensitivity of approximately 80 dB is reached at a 1-image/s acquisition rate, which allows tomography in scattering media such as biological tissues.

Microscopy, Interference↗