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Characterization of bone marrow stromal cells from multiple myeloma.

We have cultured multiple myeloma (MM) bone marrow (BM) stromal cells that are able to sustain the in vitro growth of monoclonal B-cells. Our aim was to evaluate which adhesion molecules are expressed and which extracellular matrix proteins are produced by these cells and whether they differ from the stromal cells that can be grown under the same experimental conditions from the BM of monoclonal gammopathies of undetermined significance (MGUS) and of normal donors. MM BM stromal cells that support malignant B-cell development have a striking proliferative ability that is absent in MGUS and normal donors of the same age group and are formed by four major different cell populations. Two kinds of HLA-DR+, CD10+ fibroblast-like cells can be recognized through the expression (or the lack) of alpha-smooth muscle actin isoform; further, macrophages and osteoclasts can be identified. Fibroblast-like cells that express alpha-smooth muscle actin isoform, often organized along stress fibers in a periodic fashion, may be considered as myofibroblasts. Fibroblast-like cells react strongly with antibodies to CD54 (ICAM-1), integrin beta 1, beta 3, beta 5 and some of associated alpha chains. Integrin beta 1 is diffusely exposed on the surface while beta 3 is clustered in focal contacts in association with vinculin. A still undetermined subpopulation of fibroblasts is highly positive for alpha v beta 5 that is clustered at focal contacts as shown by association with stress fiber termini and by interference reflection microscopy. A major difference between MM and normal donor BM stromal cells involves lower deposition and simpler organization of the extracellular matrix proteins (fibronectin, laminin, collagen type IV) deposited by MM fibroblast-like cells. CD14+ macrophages from MM, MGUS and normal donor BM are CD11a+ (alpha L), CD11b+ (alpha M), CD11c+ (alpha X), CD54+ (ICAM-1), CD56+ (N-CAM), beta 1 and beta 2 (CD18) integrin positive. The integrin beta 1 is diffusely expressed on the surface, while beta 2 is concentrated in podosomes. MM osteoclasts show a weak diffuse staining with CD54 and CD56 MoAbs; beta 1 integrin has a diffuse surface expression, while beta 3 integrin is concentrated in the podosomes. Normal donor osteoclasts are CD54- and the staining with CD56 is barely visible. These findings lead us to suggest that the microenvironment provided by MM BM may be significantly different from that of normal BM indicating its potential role in controlling the local proliferation and differentiation of malignant B-lineage cells.

Aged↗

A laser interferometer for sub-nanometre measurements in the cochlea.

A modification of a light microscope is described here which allows measurement of nanometre movements along the optical axis of the microscope. The light path is reflected off 10-microns-diametre small glass beads which are individually imaged with the microscope objective. The interferometer is under computer control to allow it to remain in quadrature and so maximise sensitivity. The algorithm is described. Although the techniques are applied to detection of movements of the cochlear partition, they can be used to measure sub-micron movements of any reflecting structures accessible to microscopy.

Animals↗

The organization of the cardiac ganglion of the axolotl (Ambystoma mexicanum).

The heart of the axolotl Ambystoma mexicanum was studied with histochemical methods to determine the distribution of neurons containing acetylcholine esterase, catecholamines and 5-hydroxytryptamine. The cardiac ganglion is made up of cholinergic nerve fibers and somata, and of catecholaminergic fibers. Small intensely fluorescent cells were found along blood vessels in the pericardial wall at the base of the heart, but not in the heart itself, except, in a few instances, in the region bordering the pericardial wall. Both the cholinergic and the catecholaminergic innervation of the heart were poorly developed at hatching and reached their mature state after a few months. Cholinesterase staining fibers appeared several weeks before catecholaminergic fibers. The number of postganglionic cholinergic neurons in the heart increased several-fold during the first month after hatching. Histofluorescence studies of organ cultures suggested that all the catecholamine present in the heart are of extrinsic origin. Liquid chromatography with electrochemical detection demonstrated that the dominant catecholamine in the heart is norepinephrine. No neurons containing 5-hydroxytryptamine were found.

Acetylcholinesterase↗

Mouse thymic dendritic cell subpopulations.

Mouse thymic dendritic cells (DC) have been isolated after collagenase digestion, selection of the low-density cell fraction, then depletion of T-lineage cells and other non-DC by treatment with specific monoclonal antibodies (mAb) and removal with anti-Ig-coated magnetic beads. The resulting DC preparation represented 0.1-0.2% of total thymic cells and contained 70-80% DC. Flow cytometry analysis of MHC class II (MHC II) expression by DC showed that 40% of DC expressed intermediate levels of MHC II, and 60% expressed high levels of this marker. Moreover, immunofluorescent 2-colour staining allowed the characterization of two clearly distinguishable DC subpopulations: MHC IIinter DC were CD45hi, CD44hi, HSAhi, whereas MHC IIhi DC were CD45lo, CD44lo, HSAlo. These results are discussed with regard to the functional significance of MHC IIinter and MHC IIhi DC subpopulations in the mouse thymus.

Animals↗

Selective growth of hippocampal neurites on cryostat sections of rat brain.

Dissociated rat hippocampal neurons were cultured on horizontal cryostat sections from neonatal and adult rat brain and their growth patterns visualized by brightfield and interference contrast microscopy. Cells adhered to the sections as individuals or in small clusters and grew extensive neurites. Neurites grew over all areas of neonatal sections without apparent selectivity. For adult sections, however, neurites grew almost exclusively on areas of grey matter: there was no neurite growth on areas of white matter, irrespective of the location of that white matter within the brain. The transition from the neonatal to the adult pattern of growth occurred for sections from animals aged between 14 and 21 days.

Animals↗

In vitro studies of the thyroglobulin degradation pathway: endocytosis and delivery of thyroglobulin to lysosomes, release of thyroglobulin cleavage products--iodotyrosines and iodothyronines.

UNLABELLED: Iodinated thyroglobulin stored in the thyroid follicular lumen is subjected to an internalization process and thought to be transferred into the lysosomal compartment for proteolytic cleavage and thyroid hormone release. In the present study, we have designed in vitro models to study: 1) the transfer of endocytosed thyroglobulin into lysosomes, and 2) the intracellular fate of free thyroid hormones and iodinated precursors generated by intralysosomal proteolysis of thyroglobulin. Open follicles prepared from pig thyroid tissue by collagenase treatment were used to probe the delivery of exogenous thyroglobulin to lysosomes via the differentiated apical cell membrane. Open follicles were incubated with pure [125I]thyroglobulin with or without unlabeled thyroglobulin in the presence or in the absence of chloroquine. Subcellular fractionation on a Percoll gradient showed that [125I]thyroglobulin was internalized and present in low (for the major part) and high density thyroid vesicles. In chloroquine-treated open follicles, we observed the appearance of a definite fraction of [125I]thyroglobulin in a lysosome subpopulation having the expected properties of phagolysosomes or secondary lysosomes. In contrast, in control open follicles, the amount of [125I]thyroglobulin or degradation products found in high density vesicles was lower and associated with the bulk of lysosomes, i.e., primary lysosomes. The content in thyroglobulin and degradation products of lysosomes at steady-state was analyzed by Western blot using polyclonal anti-pig thyroglobulin antibodies. Under reducing conditions, immunoreactive thyroglobulin species correspond to polypeptides with molecular weights ranging from 130,000 to less than 20,000. The presence of free thyroid hormones and iodotyrosines inside lysosomes and their intracellular fate was studied in dispersed thyroid cells labeled with [125I]iodide. Neo-iodinated [125I]thyroglobulin gave rise to free [125I]T4 which was secreted into the medium. In addition to released [125I]T4, a fraction of free [125I]T4 was identified inside the cells. Lysosomes isolated from dispersed thyroid cells did not contain significant amounts of free [125I]T4. The free intracellular [125I]T4 fraction seems to represent an intermediate 'hormonal pool' between thyroglobulin-bound T4 and secreted T4. Evidence for such a precursor-product relationship was obtained from pulse-chase experiments. IN CONCLUSION: 1) open thyroid follicles have the ability to internalize thyroglobulin by a mechanism of limited capacity and to address the endocytosed ligand to lysosomes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

The cGMP-gated channel of rod outer segments is not localized in bipolar cells of the mammalian retina.

It has recently been suggested (Nature, 346 (1990) 269-271) that ON-bipolar cells express the same biochemical cascade and guanosine 3',5'-cyclic monophosphate (cGMP)-gated cation channel as rod outer segments. An antibody directed against the cGMP-gated channel of bovine rod outer segments was applied to cryostat sections of rat and cat retinae. No immunocytochemical labelling was found in bipolar cells. Therefore, if those cells express a cGMP-gated channel, it must be immunologically different to the 63 kDa protein constituting the cGMP-gated channel of the outer segment.

Animals↗

The spectral signal-to-noise ratio resolution criterion: computational efficiency and statistical precision.

This note describes a practical improvement in the computational efficiency of the spectral signal-to-noise ratio (SSNR) resolution criterion for correlation-averaged images. The total set of N images is randomly partitioned into ng subsets, each subset is separately averaged, and a reduced form of the SSNR is computed from these average images. In general, larger values of ng achieve lower statistical uncertainty, while smaller values of ng are computationally more expedient. It is shown that, for negatively stained data, a judicious compromise is achieved with 10 less than or equal to ng less than or equal to 20, regardless of how large N may be.

Microscopy, Interference↗

Dielectric monitoring of rouleaux formation in human whole blood: a feasibility study.

In search of a method for detecting rouleaux formation in vitro, we studied the dielectric behavior of human blood under both agitated and stationary conditions. Among the parameters examined, relative permittivity ('dielectric constant') at 50-100 kHz was found to be a suitable measure of rouleaux growth, which has been difficult to quantify through conventional optical approaches. The electrical method presented here appears applicable to the kinetic analysis of rouleaux formation in undiluted whole blood.

Electric Conductivity↗

Somatostatin is contained in and released from cholinergic nerves in the heart of the toad Bufo marinus.

The heart of the toad Bufo marinus contained a substance with somatostatin-like immunoreactivity which eluted with somatostatin on reverse phase high pressure liquid chromatography. Immunoreactivity to somatostatin was localised histochemically to nerve fibers in muscle bundles of the sinus venosus, atria and ventricles and to nerve cell bodies in the sinus venosus and inter-atrial septum. Nerve cell bodies were localised both by interference contrast microscopy and immunohistochemistry; all detectable intracardiac neurons were immunoreactive. Synthetic somatostatin inhibited the rate and force of beat of atrial preparations, but did not affect the driven ventricle. Vagal stimulation caused inhibition of all cardiac chambers. After muscarinic blockade with hyoscine, vagal stimulation with 3 Hz or more still caused inhibition of the pacemaker and atrium, but not of the ventricle. The hyoscine-resistant vagal effects were diminished by about 60% after induction of tachyphylaxis to somatostatin. When when the vagus nerves were stimulated intermittently for 1 h at 10 Hz, in the presence or absence of hyoscine, the effect of somatostatin was reduced by about 60%. It is concluded that the cholinergic postganglionic neurons of the cardiac vagus contain somatostatin. When the vagus is stimulated at 3 Hz or more, the neurons release sufficient somatostatin to inhibit the pacemaker and atrial muscle.

Animals↗

Actomyosin organisation for adhesion, spreading, growth and movement in chick fibroblasts.

Examination of the actomyosin structures and their relation to adhesion, movement and growth in the first fibroblasts migrating from chick heart explants shows striking differences with fibroblasts adapted to grow in culture. The latter have focal adhesions which seem to immobilize them for anchorage-dependent growth, rather than facilitate their movement. The fibroblasts specialized for movement from the explants, though equally well spread, make contact with substratum through extensive areas of relatively unspecialized membrane, have less well developed stress fibres and a low growth rate.

Actomyosin↗

Loss of focal contacts accompanies the density dependent inhibition of cell growth.

The formation of focal contacts by fibroblast-like 3T3 and 9390 cells and endothelial XTH-2 cells was examined with the use of reflection interference contrast microscopy at different cell densities. It was found that cells density-inhibited in growth loose all focal contacts with the substratum. This observation explains the disappearance of stress fibers in density-inhibited cells, and also offers an explanation for the mechanism of density-dependent inhibition of proliferation of anchorage-dependent cells.

Animals↗

Competition of contacting heterotypic epithelial sheets for the territory in culture.

Co-cultured epithelial cells of various lines formed mixed cohesive sheets. Contact interactions of different cells in the sheets were studied by phase contrast and interference reflection microscopy, time-lapse microcinematography, transmission and scanning electron microscopy. It was found that heterotypic cells in the combined sheets were locked together by the complexes of specialized contacts and were metabolically coupled, as shown by the dye transfer. At the same time heterotypic cells continued to extend pseudopods at the contacting lateral surfaces and to attach these pseudopodies to substratum. Due to competition of pseudopod attachments one group of cells in the sheet often progressively pushed another group from the substratum. This competition of cells for substratum may provide an important mechanism for morphogenetic reorganisation of epithelial sheets and of other groups of interacting cells in vivo.

Animals↗

Videomicroscopy of acute brain slices from amygdala and hippocampus.

Video-enhanced contrast, differential-interference contrast microscopy (VEC-DICM) was used to visualize the cytoarchitecture and subcellular neuronal structure of acute brain slices from rat hippocampus and amygdala. Even at low-power magnification, the VEC-DICM system vastly improved our ability to visualize and examine the gross organization of the tissue. With medium-power magnification, the neuronal somata and proximal dendrites were clearly visible. With high-power magnification, some of the subcellular details could be clearly discerned--including cell nuclei, cell nucleoli, fine dendritic processes, and varicosities that may be synaptic expansions. We conclude that improved optical techniques should be valuable to cellular neurobiologists interested in structure-function relationships in brain slices.

Aging↗

Amplified expression constructs for human tissue-type plasminogen activator in Chinese hamster ovary cells: instability in the absence of selective pressure.

By linking an expression cassette for human tissue-type plasminogen activator (t-PA) to an amplifiable marker gene, its introduction into Chinese hamster ovary dhfr- cells and subsequent amplification with methotrexate, we have generated cell lines that overproduce the heterologous protein and contain 300-1100 copies of the expression constructs integrated into the hamster genome. We present a detailed investigation of the fate of amplified sequences in the presence and absence of selective pressure by parallel examination of three producer cell lines with respect to relevant parameters. These include the determination of t-PA production upon continuous propagation in culture, the genomic organization of the integrated expression constructs by Southern blotting, and the localization of homogeneously staining regions by in-situ hybridization with biotinylated probes and visualization by interference reflection microscopy. We conclude that in the three cell lines examined, the decrease in production of t-PA in the absence of methotrexate selection is accompanied by decreases in the number of integrated expression constructs and the size of the amplified regions, whereas all these parameters are stable when selective pressure is maintained. The instability is probably due to the head-to-tail mode of integration of the expression constructs in the hamster genome, which increases the frequency of homologous recombination between the integrated plasmids in recombination-proficient cells in the absence of selective pressure.

Animals↗

The surface morphology of the avian tectorial membrane.

The structure of the tectorial membrane of the chick was evaluated by scanning electron microscopy (SEM), using standard techniques, and, for the first time, by studying unfixed tectorial membranes with video-enhanced light microscopy techniques (AVEC-DIC). The SEM pictures show a widely varying morphology, ranging from a fully perforated tectorial membrane to a completely closed upper boundary, with a smooth surface. Based on several indicators, it is concluded that the latter presents the more natural state. This was confirmed by the results of the AVEC-DIC technique, which show a highly homogeneous structure. In contrast to the bulk of the tectorial membrane, its lower surface shows discrete structures, especially regularly oriented fibril bundles.

Animals↗