Interference contrast microscopy of living human oocytes.
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Cell Membrane↗
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Nomarski differential interference contrast microscopy and scanning electron microscopy of tissue sections and fibroblast cell culture monolayers.
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Aorta↗
[Interference contrast microscopy of the anterior eye segment in contact lens wearers].
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Anterior Eye Segment↗
Knobs on the surface of erythrocytes infected with Plasmodium falciparum: visualization by video-enhanced, differential interference contrast microscopy.
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Animals↗
[Investigation of initial stage of fibroblast attachment by means of interference reflection microscopy].
The initial cell--substrate contach, arising before the beginning of fibroblast spreading is formed from the multitude of discrete microcontacts (MC). The number of these MC increases monotonously and at room temperature 20 degrees C reaches the constant maximum value three hours after the cell settling from the suspension. An increase of MC number is accompanied by two phenomena: 1) expansion of the MC zone from the cell pole to periphery; 2) the following formation of the central continuous contact as a consequence of the fusion of the adjacent discrete MC. The zone of continuous contact expands also from the pole to periphery. The quantitative characteristics of this process are presented and the model of initial attachment as the successive attachments of the cell surface projections are discussed.
Animals↗
Differential interference contrast microscopy of human atherosclerotic lesions.
Differential interference contrast microscopy was employed to study sections of human cerebral arteries and aorta. When this procedure was used to observe 0.5-mu-thick sections of plastic-embedded arteries, images were obtained with greater definition of detail than corresponding micrographs of the area using conventional bright-field microscopy. Since structures with different refractive indices are shown in geometric relief from one another, an apparent three-dimensional image is seen, which, together with the theoretically higher resolution and contrast achieved by this technic, gives an image with definition between conventional light and low magnification electron microscopy. The morphology of fatty streak lesions with aggregates of round and elongated lipid-filled cells was demonstrated, and clear images of different forms and sizes of intracellular lipid droplets were illustrated at magnifications around 1000. In atheromatous plaques, intra- and extracellular lipid droplets were observed, some with partially extracted cores or surfaces, as were amorphous lipid droplets coalescing with crystals whose centers had been extracted. We conclude that this optical accessory to a research photomicroscope is a valuable supplemental tool in studies on the morphology of atherosclerotic lesions.
Aorta↗
The Bacteroides glycocalyx as visualized by differential interference contrast microscopy.
The glycocalyx of eight strains representing six species of Bacteroides was examined by differential interference contrast microscopy. Wet mounts in India ink were prepared from bacteria cultured in broth and on an agar medium; the wet mounts were observed by phase-contrast microscopy and differential interference contrast microscopy. With differential interference contrast microscopy, all bacteria demonstrated a glycocalyx, which included capsules surrounding single cells and microcolonies, strands of glycocalyx connecting cells and microcolonies, detached slime, and solid masses of glycocalyx in which innumerable bacteria were enmeshed. Bacteria showed comparable amounts of glycocalyx by visual observation with differential interference contrast microscopy whether grown on plates or in broth. Serial transfers of cultures did not diminish the amount of glycocalyx. Differential interference contrast microscopy proved to be a superior method to phase contrast for examining wet preparations of Bacteroides.
Bacteroides↗
Microtubule-associated movement of mitochondria and small particles in Acanthamoeba castellanii.
Using video-enhanced differential interference microscopy and digital image processing, we have observed organelle motility in Acanthamoeba castellanii. In amoebae taken from cultures in rapid growth phase, mitochondria and small particles moved over distances of several microns and at an average velocity of approximately 2 microns/s. Mitochondrial motility was verified by intensified fluorescence microscopy of cells that were labeled in vivo with the DNA-binding dye DAPI or the mitochondria-specific dye MitoTracker. We further studied the role of microtubules (MTs) in the translocation of cell organelles. Double-labeling of fixed cells with mitochondrial markers (anti-F1 beta antibody, MitoTracker) and cytoskeletal markers (anti-tubulin antibody, rhodamine-phalloidin) demonstrate that the mitochondria colocalize with MTs in the subcortical cell area and are excluded from the F-actin-rich cell cortex. Colchicine treatment resulted in an almost complete depolymerization of MTs and an inhibition of organelle motility. Moreover, we have directly visualized MTs in vivo in flattened amoebae. Mitochondria and small particles moved along the MTs in a bidirectional mode at an average velocity of approximately 1 micron/s. We conclude that the observed movement of mitochondria and small particles in Acanthamoeba castellanii mainly occurs via microtubules and associated motor proteins.
Acanthamoeba↗
Stringent requirement for Ca2+ in the removal of Z-lines and alpha-actinin from isolated myofibrils by Ca2+-activated neutral proteinase.
Treatment of isolated myofibrils with Ca2+-activated neutral proteinase (CANP) results in specific removal of Z-line and of alpha-actinin. To investigate the ionic requirement for these processes, we measured Z-line removal by phase-contrast and interference microscopy and alpha-actinin removal by sodium dodecyl sulphate/polyacrylamide-gel electrophoretic analysis of myofibrillar proteins. The proteolytic digestion of native purified proteins was measured directly on polyacrylamide gels and by the fluorescamine technique. We found that the removal of Z-line and alpha-actinin as well as the release of proteolytic degradation products from isolated myofibrils by CANP occur only in the presence of Ca2+; Sr2+, Ba2+, Mn2+, Mg2+, Co2+ and Zn2+ are all ineffective. In contrast with this stringent requirement for Ca2+, the proteolytic activity of CANP measured with denatured casein, native and denatured haemoglobin, native actin and tropomyosin also occurs in the presence of other bivalent cations, in the following order: Ca2+ greater than Sr2+ greater than Ba2+. These data suggest that only Ca2+ can produce the conformational change in myofibrils that renders them susceptible to the action of CANP, whereas its proteolytic activity is stimulated by several bivalent ions.
Actinin↗
Amoeboid locomotion of Acanthamoeba castellanii with special reference to cell-substratum interactions.
The amoeboid locomotion of Acanthamoeba castellanii has been studied by observation of individual cells moving on a planar glass substratum. Cell-substratum interactions involved in traction have been observed by reflexion interference microscopy. A variable part of the ventral surface of A. castellanii formed a protean platform, the 'associated contact', from which filopodia were subtended; these established stable, focal adhesions (approximately 0.4 micron diameter) on the substratum beneath. Surprisingly, acanthopodia, a prominent feature of this protozoon, did not play an obvious role in traction. The dimensions of the cell-substratum gap in the associated contact could be modulated by the concentration of ambient electrolyte. Dilution of electrolyte from 50 mM-KC1 to 2mM resulted in (i) an increase in the cell-substratum gap, (ii) a marked decrease in cell motility, (iii) reduced cell adhesion to glass.
Adhesiveness↗
T-mycoplasmas: a study of the morphology, ultrastructure and mode of division of some human strains.
The morphology of 10 strains of T-mycoplasma was studied in wet preparations of viable cells by darkfield, phase-contrast and interference microscopy, and in fixed preparations by various techniques of electron microscopy. Mycoplasma-like artefacts in the horse-serum component of the medium were eliminated by filtration. All 10 strains were similar. Individual cells were spherical, 0-25-1-0 mum in size, with a bounding trilaminar membrane, 10 nm thick and containing 7-5-12-5-nm particles, and a layer of pilus-like projections, 5-8 nm long, on the outer surface. A possible capsular matrix was observed only by the pseudoreplica technique. The cells contained 12-15-nm ribosomes, nuclear fibroids 7-5-9 nm wide, and vacuoles. During replication, the cell elongated slightly and the ribsomes migrated to the ends of the cell leaving a ribosome-free area into which the bounding membrane invaginated to form a bud. The bud eventually separated by completion of the process of invagination; a cross-septum did not form. Usually only a single bud developed but sometimes two appeared simultaneously.
Cell Membrane↗
A method of permanently mounting biological tissue cleared in Herr's four-and-a-half clearing fluid.
A technique which should be generally applicable for preparing permanent mounts of tissue cleared in Herr's four-and-a-half clearing fluid is described. This technique involves transferring plant or animal tissues through a series of solutions consisting of Pienarr's fixative, Herr's clearing fluid, chloral hydrate, acetone and finally polyester resin for mounting. Material prepared using this method is exceptionally transparent and well preserved, and is suitable for either phase contrast or Nomarski interference microscopy.
Chloral Hydrate↗
[Changes in the structural heterogeneity of surviving nerve cells].
Heterogeneity in distribution of the dry substance mass (protein) in normal and reactively altered isolated neurons has been studied in vital investigations by means of interference microscopy and microspectrophotometry. It has been demonstrated that at a reactive reconstruction neuroplasmic substances undergo rearrangement, resulting in increased heterogeneity. In peripheral juxtamembranous parts neuroplasmic colloids are divided separating the hydrated zone with a low content of protein and forming made that an increasing adhesion of the juxtamembranous proteins could result in their aggregation with protein complexes of the transmembranous ionic canals, that affects the specific electrogencic function of the neuron.
Animals↗
[Morphology of the Meibomian lipid film. Results of Brewster angle microscopy].
Meibomian lipid layers were studied in the anterior mirror area by reflecting microscopy and interference microscopy. Using these techniques, it was not possible to correlate the biophysical and morphological data. Brewster angle microscopy provides direct observation of the spread Meibomian lipid layer with simultaneous registration of the surface pressure. It is based on the fact that He-Ne laser light, which is incident at a water surface under the Brewster angle, does not reflect visible light. After spreading of a lipid film, the angle of the incident light beam varies, causing reflection of light. The Meibomian lipid layer was studied in a Langmuir-type trough. At 5.0 mN/m the lipid layers are homogeneous and mobile, consisting of areas of higher and lower reflectivity. In patients with meibomitis the films are inhomogeneous and immobile. The thickness of the areas of lower reflectivity is 2 nm, the high reflectivity lipids are 8-10 nm thick. According to these results, Meibomian gland secretion can form monolayers under in vitro conditions.
Blepharitis↗
Long-term observation of cultured cells by interference-reflection microscopy: near-infrared illumination and Y-contrast image processing.
Interference-reflection microscopy (IRM) is the only method presently available with which to visualize cell-substratum adhesions in living tissue culture cells continuously for long periods of time without the use of fluorescent markers (Curtis: J. Cell Biol. 20:199-215, 1964; Izzard and Lochner: J. Cell Sci. 21:129-159, 1976). This method utilizes approximately 1% of the incident illumination to produce the IRM image (Verschueren: J. Cell Sci. 75:279-301, 1985) and so far has required the use of high-intensity light sources in the visible spectral range (400-800 nm). Unfortunately, visible light of this intensity and spectral range induces marked changes in the behavior and morphology of motile fibroblasts, including cessation of locomotion. In contrast, the present paper reports that continuous observations of live cells in IRM for periods of up to 8 hours are possible if the illuminating light is in the red to near-infrared range (650-950 nm) and without any observable change in normal cell morphology or behavior. In addition, we describe how the technique of Y-contrast image processing can be applied to IRM images to create a three-dimensional image of the ventral cell surface topography.
Animals↗
Application of video-enhanced interference reflection microscopy to the study of platelet-surface interactions.
Video-enhanced interference reflection microscopy (VEIRM) was used to examine contact sites between the ventral membrane of intact platelets and underlying surfaces. It was observed that the ventral membrane in the central granulomere region of fully spread platelets was separated from the surface, while the membrane in other regions was mostly in close contact. The VEIRM image of intact platelets was compared with the video-intensified fluorescence microscopic (VIFM) image of cytoskeletal structures labeled with rhodamine-phalloidin. The VEIRM was also used to visualize the cytoskeletal structures of platelets spread on glass surfaces. Platelets were treated with Triton X-100, glutaraldehyde and acetic acid in sequence. This method was used to follow the sequence of cytoskeletal reorganization of platelets after surface-induced activation. In addition, the effects of albumin and fibrinogen on the cytoskeletal reorganization of spreading platelets were investigated. On fibrinogen-coated surfaces, platelets developed extensive inner filamentous zones which encircled the central granulomere region. In the presence of albumin, however, platelet inner filamentous zones were very poorly developed.
Actins↗
Use of differential interference contrast microscopy to determine cell renewal times in mouse intestine.
Pieces of mouse mid jejunum have been cleared in glycerin and examined by differential interference contrast microscopy at low powers of magnification. The position and number of crypts and villi can be determined in the same specimen using this technique. The calculated value for crypt/villus ratio 4.53 +/- 0.99 (mean +/- SD) is less than a previously published value obtained using indirect techniques. A revised estimate of cell renewal time, based on this newly determined value for crypt/villus ratio, is 45 h. This agrees with earlier estimates derived from entirely different methods of analysis. The general usefulness of this form of light microscopy in helping one appreciate some three-dimensional problems in mucosal architecture is emphasized.
Animals↗
[The application of interference contrast microscopy in biology (author's transl)].
Like phase contrast, Nomarski interference contrast microscopy can be used to examine unstained specimens in biology and medicine. The properties of both contrast enhancement techniques are illustrated by various examples. The phase contrast method is especially suited for thin specimens with small differences in refractive index, whereas the interference contrast method supplies good results even of thick specimens. Interference contrast is a valuable supplement to the phase contrast method, and expands the application of microscopy in biology
Animals↗