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PubMed1992

Observations on the turkey oviductal sperm-storage tubule using differential interference contrast microscopy.

Squash preparations of unfixed, uterovaginal junction mucosae revealed that openings to sperm-storage tubules were round or slit-like and were surrounded by either cilia, which were part of the uterovaginal junction surface epithelium, or nonciliated cells resembling the sperm-storage tubule epithelium. By focusing on different levels of the sperm-storage tubule (optical sectioning), connective tissue fibres and cells between individual sperm-storage tubules, epithelium and lumen of sperm-storage tubules containing resident spermatozoa were observed. An optical section through the sperm-storage tubule epithelium revealed basal nuclei and associated nucleoli, and refractile supranuclear lipid droplets. Luminal spermatozoa were distributed primarily in the distal third of the sperm-storage tubule and nearly always formed a tight bundle at its base. These spermatozoa were often observed slowly and synchronously oscillating. In two-thirds of the 30-week-old, non-photostimulated hens, sperm-storage tubules were fully formed. In contrast, the remaining hens possessed bud-like surface invaginations lacking discernible lumina. It was concluded that differential interference contrast microscopy offers better spatial and optical resolution of the sperm-storage tubule than other modes of light microscopy.

Animals↗

PubMed1976

Observations of the microcirculatory bed in rat mesocecum using differential interference constrast microscopy in vivo and electron microscopy.

The microvascular bed of the rat mesocecum has been examined in vivo using differential interference (Nomarski) optics and subsequently by electron microscopy. The preferential channel, from terminal arteriole to collecting venule, has been examined. In the terminal arteriolar segment the endothelial layer is covered by a continuous layer of smooth muscle cells which, in turn, are surrounded by adventitia. In the metarteriolar segment the periendothelial cells still resemble smooth muscle cells but the tunica media is discontinuous. In the distal segment periendothelial cells are more scattered and have the appearance of pericytes. There appears to be a continuous transition of the periendothelial cell layer from terminal arteriole to distal segment. Nerve endings were seeen in both the terminal arteriolar and metarteriolar segments. During contraction smooth muscle cells, oriented circumferentially, shorten and thicken. Endothelial cells appear anchored by myoendothelial junctions. Endothelial cells have filaments which show increased banding during vasoconstriction, suggesting that such cells may contract. Capillary offshoots leave the preferential channel, usually at right angles. Smooth muscle cells are oriented to form a sphincter and there are many myoendothelial junctions at the branch point. Within a short distance the capillary branch loses its periendothelial coat.

Animals↗

PubMed1983

Motility of human polymorphonuclear neutrophils: microscopic analysis of substrate adhesion and distribution of F-actin.

Directed movement of polymorphonuclear neutrophils (PMN) requires cell polarization and the orderly making and breaking of cell-substrate contacts. We compared the movement of human PMN suspended from the underside of glass coverslips to that of PMN seen in "profile" on fibers, using brightfield, differential interference contrast and reflection interference microscopy. Images were recorded on film and videotape and analyzed in real time and time lapse. The distribution of F-actin was observed with image-enhanced fluorescence microscopy after staining with NBD-phallacidin. PMN exhibited two patterns of motility. Fifteen to twenty-five percent of cells moved in a low profile gliding pattern and exhibited caudad displacement of dorsal surface folds. Most PMN made progress by cycles of partial release of the lamellipodium from the substrate and anterior advance followed by arching or rolling and lamellipodial reassociation with the substrate. Cells stimulated with bacteria, casein, or chemotactic formyl peptide rarely spread on the coverglass but waved into the medium attached only by the uropod. Eventually, many detached completely from the substrate. Cells confined to the substrate surface with overlying agarose were able to locomote when confronted with these substances. F-actin was irregularly distributed in nonpolarized suspended cells but concentrated in the lamellipodium in polarized cells. As cells arched along a substrate, F-actin accumulated in foci corresponding to the substrate-PMN interface, particularly at the uropod and retraction fibrils. Conversely, cells that were physically restricted to movement in the plane of the substrate surface by overlying agarose exhibited diffuse F-actin along the entire cell. Suspended PMN polarized with formyl peptide and incubated with Con A accumulated F-actin at the uropod. These observations suggest that both PMN locomotion and the movement of Con A binding sites involve the caudad redistribution of F-actin.

Actins↗

PubMed1990

Macrophages form circular zones of very close apposition to IgG-coated surfaces.

When phagocytes spread on surfaces coated with ligands such as IgG, they form a tight seal with the substrate. This seal excludes soluble macromolecules in the medium from the interface between the cell and substrate. In contrast, when cells spread on control surfaces that are not coated with ligands, the underside of the cell remains freely accessible to soluble proteins (Wright and Silverstein: Nature 309:359, 1984). We employed reflection-interference microscopy (RIM) to determine where the seal forms during interaction with ligand (IgG)-coated surfaces. Human monocyte-derived macrophages (MO) were plated at 37 degrees C on dinitrophenylated (DNP)-glass coverslips (control substrate), IgM anti-DNP-DNP-coated glass (control substrate), or on IgG anti-DNP-DNP-coated glass (phagocytosis-promoting substrate). Live or fixed cells were examined by RIM. Spreading on control surfaces at 37 degrees C was complete in 25 minutes, whereas spreading on IgG-coated surfaces was maximal within 15 minutes and resulted in cell-substrate contact area 1.6 X that of control cells. Within 1 h at 37 degrees C, 90% of MO that spread on IgG-coated substrates, but not on control substrates, excluded macromolecules from their underside. A minor population of cells (19%) exhibited a uniform iron gray RIM appearance indicating an even, close approach to the substrate. These cells may represent early stages of frustrated phagocytosis. In contrast to cells on control substrates, 70% of cells on IgG-coated substrates developed continuous peripheral dark rings in RIM indicative of close association with the substrate. Essentially all cells with peripheral dark rings in RIM excluded macromolecules from their underside. Enclosed within this ring was an area of greater separation between the cell membrane and the substrate, as indicated by the lighter grey of this region in RIM and by the accessibility of substrate to anti-substrate antibody when breaks in the dark ring occur. Thus, MO can create a closed compartment between plasma membrane and substrate that excludes proteins in the surrounding medium, thereby protecting substances secreted into this space from potentially inhibitory substances in the medium.

Cell Communication↗

PubMed1992

Fertilization alters the orientation of pigment granule saltations in Arbacia eggs.

Unfertilized eggs of the sea urchin Arbacia punctulata contain pigment granules distributed throughout their cytoplasm. During the first 15 minutes after fertilization, these vesicles move out to the cortex where they become firmly anchored. We have used time-lapse video differential interference microscopy to analyze the motility of these organelles in unfertilized and fertilized Arbacia eggs. Pigment granules exhibit saltatory movement in both unfertilized and fertilized eggs. Quantitation of vesicle saltations before and after fertilization demonstrates that while there is no significant difference in the speed or path-length of vesicle movement, there is a dramatic change in the orientation of these saltations. Saltations in the unfertilized egg are very non-radial and are as likely to be directed toward the cortex as away. In contrast, saltations in the fertilized egg are more radially oriented and more likely to be cortically directed. This transition must reflect underlying changes in the cellular structures necessary for pigment granule saltations. The change in the orientation of pigment granule saltations following fertilization requires both a transient increase in the cytoplasmic concentration of Ca2+ and an elevation of cytoplasmic pH. Similarly, the ability of pigment granules to adhere to the cortex requires both the transient elevation of cytoplasmic Ca2+ and the alkalinization of the cytoplasm. As the reorganization of cortical actin at fertilization is regulated by these ionic fluxes, and both movement and adhesion are sensitive to cytochalasins, we hypothesize that the alterations in directed motility and adhesion reflect underlying changes in the actin cytoskeleton.

Animals↗

PubMed1980

Changes in nucleolar dry mass of neurones of the paraventricular and supraoptic nuclei in the rat during pregnancy and lactation.

Interference microscopy was used to measure the dry mass of nucleoli in unfixed nuclei isolated from neurones of the paraventricular (PV) and supraoptic (SO) nuclei of female rats. Changes in nucleolar dry mass during pregnancy and lactation have been interpreted as reflecting changes in rates of synthesis of ribosomes and protein in these neurones. Measurements were made on a total of 6580 nucleoli from 135 rats. At the end of pregnancy nucleolar dry mass of both PV and SO neurones was increased compared with virgin female rats. Nucleolar dry mass of PV neurones but not SO neurones increased further during lactation. This change was biphasic, with a nadir at 2 weeks post partum. After day 5 post partum, nucleolar dry mass of PV and SO neurones was increased only in rats sucking pups. Adjustment of litter size to 10 or 22 to 24 pups on the first day post partum did not affect nucleolar changes in PV and SO neurones. Nucleolar changes were less when only one pup was nursed. The results are discussed in relation to oxytocin secretion induced by the suckling stimulus and the synthetic response of PV and SO neruones to increased secretion.

Animals↗

PubMed1994

In vitro exposure of a novel polyesterurethane graft to enzymes: a study of the biostability of the Vascugraft arterial prosthesis.

The biostability of the Vascugraft arterial prosthesis, a porous synthetic graft made by a novel spinning process from a unique poly(ester urethane) polymer, has been studied by means of an in vitro enzyme incubation technique. Samples of the Vascugraft were exposed to buffered solutions of collagenase and pancreatin, as well as the buffer solutions alone, for periods of up to 100 days at 37 +/- 1 degrees C. On removal and after cleaning, a number of different analytic methods, including X-ray photoelectron spectroscopy for chemical analysis (ESCA), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), size exclusion chromatography (SEC), scanning electron microscopy (SEM), interference microscopy, moisture content and contact angle measurements, were used to examine the changes in chemical structure and surface morphology of the samples. During incubation in both enzymes the molecular weight of the polyurethane appeared to decrease in the presence of enzyme but increase in the presence of buffer. Further microphase separation in the polyurethane material developed during incubation in buffer solutions. Such changes in microstructure were associated with increased surface hydrophilicity, increased moisture content and a significant improvement in the extent of order and preferred orientation of the hard segment domains within the fibres. In the sampling depth of about 5 nm, both enzymes decreased the carbonate group content at the surface of the prosthesis to as little as 40% of their original values. The results from ATR-FTIR and DSC demonstrated that this phenomenon was limited primarily to the soft segment phase. While the Vascugraft prosthesis did exhibit some limited chemical modifications on exposure to concentrated enzyme solutions, nevertheless such changes were confined to the surface layer of the polyurethane microfibres. The importance and significance of those results will be more adequately determined by in vivo investigation.

Blood Vessel Prosthesis↗

PubMed1983

Interference microscopic determination of the section thickness of different paraffin-embedded organ tissues.

With respect to the significance of the section thickness in quantitative-morphological studies the thickness of different paraffin-embedded tissue sections was determined by interference microscopy. Within the range of 3-5 microns the measurements revealed accordance of the measured section thickness with that adjusted on the sliding microtome. In the range surpassing 5 microns the section thickness obtained was in all cases lower than that adjusted on the microtome. The deviations were up to 40%.

Histological Techniques↗

PubMed1992

The direct effects of graded axonal compression on axoplasm and fast axoplasmic transport.

The direct effects of mechanical compression on axoplasm and fast axoplasmic transport were studied by video-enhanced differential interference microscopy. Single axons, isolated from the squid, were compressed with 0.5, 5, 20, or 100 gram (g) weights placed over a 1 millimeter (mm) length of axon. Brief compressions (10 seconds) at low pressures (0.5 g/mm) momentarily deformed the axon, but the axoplasm and axon returned to their normal shape and position after the pressure was removed, and no residual changes in axoplasmic structures, fast axoplasmic transport or membrane function were seen. Compressing the axon with 5-20 g/mm, however, broke the axoplasm at the site of the crush and squeezed the axoplasm out from under the compression site. Though the axoplasm usually returned to the crush site after the weight was removed and organelles continued to move in the axoplasm under the crush, the organelles failed to cross a dense line that marked the site of the rejoined axoplasm, instead they accumulated over time at the crush site. This results suggests that the blockage of fast transport at moderate compressions was due to a mechanical breakage of the axoplasm at the compression site. The plasma membrane was apparently not transected after moderate compressions (5-20 g/mm) since the resting membrane potential returned to nearly control levels after the weight was removed. Compressions with 100 g/mm, however, did break the plasma membrane as evidenced by the rapid and irreversible loss of the action potential and resting potential and the ion-dependent liquefaction of axoplasm and loss of all organelle transport at the 100 g/mm compression site. Thus, small mechanical pressure elastically deformed the axoplasm, moderate pressures mechanically broke the axoplasm, and high pressures broke the axoplasm and the plasma membrane.

Action Potentials↗

PubMed1969

The response of motor neurones to intramuscular injection of botulinum toxin.

1. The dry mass and nucleic acid content of both nerve cell bodies and their nucleoli were measured by interference microscopy and ultra-violet absorption microspectrography respectively: succinoxidase and acetylcholine hydrolase activities were also determined. Autoradiography was used to follow synthesis of deoxyribonucleic acid (DNA) by glial cells, and to follow nucleic acid and protein metabolism in muscle fibres.2. After injection of botulinum toxin the synthesis of ribosomal RNA by the neurone followed closely the pattern found after axotomy.3. After injection of toxin neuronal dry mass increased before the rate of ribosomal RNA synthesis was raised. This early increase, which was not due to increased protein synthesis, probably represents a ;damming back' of proteins within the nerve cell body.4. After injection of toxin no local accumulation of microglial cells synthesizing DNA was found around the affected neurones: it is suggested that this reflects the intact system for intra-axonal transport under these conditions.5. The affected muscles show increased nucleic acid and protein synthesis.6. It is suggested that the results obtained indicate that membrane expansion or synthesis which occurs both in muscle and in neurone under these circumstances is the factor responsible for inducing directly or indirectly the changes found in nucleic acid metabolism after injection of botulinum toxin and after axotomy.

Animals↗

PubMed1972

Some quantitative observations upon the responses of neuroglial cells which follow axotomy of adjacent neurones.

1. The dry mass and nucleic acid content of freshly isolated neuroglial cells and of their nucleoli were measured by interference microscopy and ultraviolet absorption microspectrography. The incorporation of tritiated nucleosides and of an amino acid was followed autoradiographically.2. After hypoglossal axotomy in the adult rat hypertrophy of astrocytes of the hypoglossal nucleus occurred in a biphasic manner. The first phase lasted from days 1-10 and was accompanied by a small degree of astrocytic hyperplasia, and the second from days 20-80. Hypertrophy of oligodendrocytes accompanied the second phase of the astrocytic response.3. When severed axons failed to reinnervate denervated muscle, the second phase of the astrocytic response was markedly reduced and the hypertrophy of oligodendrocytes did not occur.4. If the severed axons re-innervated denervated muscle after a controlled delay, the second phase of the astrocytic response and the oligodendroglial hypertrophy was also delayed.5. Injection of botulinum toxin into the tongue caused changes in astrocytes and oligondendrocytes closely resembling those found after axotomy.6. Transient astrocytic hypertrophy occurred in the uninjured right hypoglossal nucleus, and had a different time course to the changes occurring on the injured side.7. The results are discussed in relation to changes in the metabolism and to alterations in the dendritic fields of injured neurones, previously measured in these circumstances.

Animals↗

PubMed1966

Structure and organization of the living mitotic spindle of Haemanthus endosperm.

New details of mitotic spindle structures in the endosperm of Haemanthus katherinae (Bak) have been demonstrated by differential interference microscopy. Spindle fibers are clearly seen in the living spindle extending from the kinetochores to the polar region. Individual spindle fibers consist of a bundle of smaller filaments which diverge slightly from the kinetochore and intermingle with filaments from other spindle fibers as they approach the polar region. The degree of intermingling increases during metaphase and anaphase. The chromosomes stop moving when the spindle fibers are still 5 to 10microns long; then the fibers disappear. These observations explain some aspects of spindle movements which were difficult to reconcile with earlier concepts of spindle organization.

Cell Division↗

PubMed1982

Refractive index of the fly rhabdomere.

The refractive index and the diameter of the fly rhabdomere were determined by comparing the experimental results derived from interference microscopy with the results of theoretical study on the scattering of plane waves by a homogeneous, isotropic cylindrical dielectric rod. It was found that the refractive index of the isolated rhabdomere of Calliphora erythrocephala is 1.363 +/- 0.003 in an area of the rhabdomere where its diameter is calculated to be 1.32 +/- 0.04 micrometers.

Animals↗

PubMed1980

Fibrous waveforms or crimp in surface and subsurface layers of hyaline cartilage maintained in its wet functional condition.

The structural features of hyaline cartilage maintained in its wet functional condition have been examined using the technique of Nomarski interference microscopy. The collagenous arrays and associated chondrocytes in both the extreme superficial layers and in the deeper subsurface zones were satisfactorily imaged with this technique. Most significantly the collagen fibers were observed to possess a geometric waveform or "crimp" of varying acuteness and the role of this crimp is discussed in relation to the mechanical and biological function of the tissue. It is clear from these wet tissue studies that the fibrous layout in hyaline cartilage is considerably more "disciplined" than has been previously recognized from morphological data obtained using more indirect experimental techniques involving prepared histological sections or scanning and transmission electron microscopy.

Animals↗

PubMed1992

A new morphological classification of urinary erythrocytes for differential diagnosis of glomerular hematuria.

A new morphological classification of urinary erythrocytes was instituted in order to differentiate glomerular from urological hematuria. One hundred and thirteen hematuric patients including 73 glomerular and 40 urological disease patients were examined. The former group consisted of IgA nephropathy (n = 45), lupus nephritis (6), membrano-proliferative glomerulonephritis (5), non-IgA mesangial proliferative glomerulonephritis (4), Henoch-Schoenlein purpura nephritis (4), membranous nephropathy (4), endocapillary proliferative glomerulonephritis (3), and minimal change nephrotic syndrome (2). The latter group included bladder cancer (n = 15), renal calculi (15), prostate cancer (3), urethral cancer (1) and post-transurethral resection (6). In each urine sample, 100 urinary erythrocytes were observed under differential interference microscopy and classified into 10 concretely defined shapes (5 "glomerular" and 5 "urological" shapes) and unclassified shapes. Using percentage of "glomerular" shape erythrocytes and setting the cut-off at 15%, 90.4% of sensitivity and 97.5% of specificity for the diagnosis of glomerular disease were obtained. When percentage of one specific shape (G1), [i.e. doughnut-like cell with one or more blebs] was used at a cut-off of 1%, sensitivity and specificity were 89.0% and 95.0% respectively. These results were satisfactory as compared with most previous reports. Moreover, our classification is so concrete that it is more objective, accurate, and easily understandable, even for beginners. Distinct shape "G1" is particularly important for morphological investigation of hematuria.

Diagnosis, Differential↗

PubMed1975

Encephalitis in suckling rats induced with rat cytomegalovirus.

Encephalitis has been induced in suckling rats by intracerebral inoculation of rat cytomegalovirus. This agent has been carried through five animal passages without changes in virulence. Principal sites of viral attack were the meninges and ependyma with limited parenchymal invasion from these sites. Characteristic features of cytomegalovirus disease observed included cytomegaly, formation of nuclear and cytoplasmic inclusions, and polykaryocytosis. Fundamental tinctorial and optical differences were noted between nuclear and cytoplasmic inclusions, the latter being characterized by their phloxinophilia which contrasts with the hematoxylinophilia of nuclear inclusions. Differential interference microscopy improved visualization and photography of cytoplasmic inclusions. Hydrocephalus developed in a few of the animals and was studied in late phases of the disease.

Animals↗

PubMed1989

Cytophotometric and interference microscopic investigations in carcinomas of the oral cavity.

The DNA content and the nuclear dry mass of 18 keratinized squamous cell carcinomas and their metastases, of one adamantinoma recurrence and two adenoid cystic carcinomas of the oral cavity were determined in comparison to the normal buccal mucosa using Feulgen scanning cytophotometry and interference microscopy. The squamous cell carcinomas could be classified into five groups based on their DNA distribution pattern. The nuclear dry mass and its variation were found to be different from the normal mucosal epithelial cells in all cases. No differences could be found between diploid cells and cells of a higher degree of ploidy. Therefore the relation of nuclear dry mass and DNA content appeared to be lower in cells of higher DNA ploidy. This fact is discussed in relation to the underlying molecular biological processes. This leads to the conclusion that the increase of the nuclear dry mass (mainly protein) precedes the increase of the DNA content. The combined measurement of DNA content and nuclear dry mass allows a better characterization of malignancy than each of the nuclear components measured alone. The difference of nuclear dry mass between malignant cells and normal mucosal epithelial cells is more sensitive than the deviation of the DNA content owing to the fact that malignant tumours with normal DNA content exist.

Carcinoma, Squamous Cell↗

PubMed1984

Microfilament distribution and adhesion patterns in cultured cells after glutaraldehyde-formaldehyde fixation.

To study the relationship between microfilament distribution and adhesion patterns in the same cultured cell, we have employed a simple glutaraldehyde-formaldehyde fixation technique followed by permeabilization of the cells in buffered Triton X-100. This method gives an excellent preservation of cellular morphology in general and of adhesion patterns in particular for examination with surface reflection interference microscopy. It also permits the concomitant use of the actin-specific fluorescent probe NBD-phallacidin to visualize the distribution of microfilaments.

Cell Adhesion↗