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At least 181 records · Page 10Linked to original sources

PubMed2005

Variable incidence angle fluorescence interference contrast microscopy for z-imaging single objects.

Surface-generated structured illumination microscopies interrogate the position of fluorescently labeled objects near surfaces with nanometer resolution along the z axis. However, these techniques are either experimentally cumbersome or applicable to a limited set of experimental systems. We present a new type of surface-generated structured illumination fluorescence microscopy, variable incidence angle fluorescence interference contrast microscopy (VIA-FLIC), in which the fluorescent sample is assembled above a reflective Si surface and the incidence angle of excitation light is varied by placing annular photomasks with different radii in the aperture diaphragm plane of the microscope. The variation in incidence angle alters the interference pattern of excitation light, and hence the intensity of detected fluorescence. Quantitative VIA-FLIC is tested by using a set of fluorophore-containing supported membranes separated from the Si surface by SiO2 layers of variable thicknesses. The resulting fluorescence intensity versus incidence angle curves depends on the separation from the Si surface and when fit with an appropriate model yield precise SiO2 thicknesses that are accurate with respect to the known SiO2 thicknesses. Since only a simple modification to a standard epifluorescence microscope is required, VIA-FLIC offers a versatile method to produce z-reconstructions with high resolution for a wide range of biological systems.

Algorithms↗

PubMed2006

Real-time dynamics of label-free single mast cell granules revealed by differential interference contrast microscopy.

We demonstrate the capability of differential interference contrast (DIC) microscopy as a simple and useful tool for studying cellular events without fluorescence labeling. By coupling an advanced DIC microscope to a computer-controlled motorized vertical stage and a high-speed, high-resolution CCD camera, real-time three-dimensional monitoring is possible in a high-throughput manner. The performance among three modes of microscopy, bright-field, dark-field and DIC, in terms of horizontal resolving power and vertical sectioning was investigated. As a model, exocytosis of rat peritoneal mast cells was recorded on the subsecond time scale. Three-dimensional tracking of granules during degranulation was achieved and granule-granule fusion before plasma membrane fusion was recorded.

Animals↗

PubMed2004

Absolute interfacial distance measurements by dual-wavelength reflection interference contrast microscopy.

Dual-wavelength reflection interference contrast microscopy (DW-RICM) is established as a microinterferometric technique to measure absolute optical distances between transparent planar substrates and hard or soft surfaces such as colloidal beads or artificial and biological membranes, which hover over the substrate. In combination with a fast image processing algorithm the technique was applied to analyze the trajectories of colloidal beads sedimenting under gravity. As the beads approach the surface of the substrate, they slow down because of hydrodynamic coupling of the bead motion to the substrate. The effective surface friction coefficients were measured as a function of the absolute distance of the beads from the surface. The height dependence of the friction coefficient was found to be in quantitative agreement with previous theoretical predictions. Furthermore, we demonstrate that the DW-RICM technique allows the determination of the height of membranes above substrates and the amplitude and direction of height fluctuations. Without any further need to label the membrane the unambiguous reconstruction of the surface profile of soft surfaces is possible.

Journal Article↗

PubMed1995

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↗

PubMed1983

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↗

PubMed1984

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↗

PubMed1975

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↗

PubMed2002

Properties of erythrocyte light refraction in diabetic patients.

Since hyperglycaemia changes the erythrocyte cell membrane fluidity and impairs cell deformity, our goal was to characterize hemoglobin and red blood cell (RBC) light refractive property changes in diabetic patients. Microscopic investigation was carried out on intact and fixed RBCs. To determine the refractive index (RI): smears of peripheral blood were air dried and fixed for 3 min in methanol. Mixtures of polyvinylpyrolidine and buffer of different pH (1:1) were used as embedding media. Intact RBCs were mixed with a buffered embedding medium, placed on a slide and overlaid with a coverslip. Interference microscopy was used for RI measurements at 18 different pH (pH=2-13). The results showed that curves of the RI of diabetic patients and of a control group were of similar configuration, with one branch in the acidic portion of the pH scale, a maximum and two minima in the neutral (middle) portion, and one branch in the alkaline portion. The curves of the individuals from the control group overlapped each other. To the contrary, the curves of the diabetic patients were not uniform in the neutral portion and the alkaline portion. The curves of the diabetic patients in the neutral zone were shifted towards the alkaline end of the pH scale, and the RBC RI curves were lower in comparison to the control curves. The center maximum of the curves of diabetic patients corresponded to pH=6.6 whereas the central maximum of the control group curves was at pH=6.2-6.8. Contrary to in the diabetic group, intact RBC RI curves in the control group revealed only one significantly different minimum at pH of 7.2 in the neutral zone. Using this method it is possible to show phenotypic differences between uniform type intact and fixed cells, erythrocytes of diabetic patients and of healthy donors.

Blood Glucose↗

PubMed1976

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↗

PubMed1981

[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↗

PubMed1995

[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↗

PubMed1989

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↗

PubMed1989

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↗

PubMed1980

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↗

PubMed1975

[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↗

PubMedDate not supplied

Immediate diagnosis in vaginal cytology using interference contrast microscopy (Nomarski).

Studying fresh aspiration material from the posterior fornix and cervix, by Interference Contrast Microscopy (Nomarski) is a good method of cytologic examination. It is shown how most cell types can be observed, just as they can be by the classical Papanicolaou staining. Normal and abnormal, even dysplastic and malignant cells can be recognized. This method is also very useful for identifying parasites, fungi and bacteria, by morphology and active movements. While encoraging the use of this method, it is advisable to compare the results with slides examined later by the Papanicolaou technique, for maximum safety of the patient.

Bacteria↗

PubMed1964

THE MECHANISM OF ADHESION OF CELLS TO GLASS. A STUDY BY INTERFERENCE REFLECTION MICROSCOPY.

An optical technique for measuring the thickness of thin films has been adapted and evaluated for studying the structure of the adhesion of cells to glass in tissue culture. This technique, which is termed interference reflection microscopy, has been used to study embryonic chick heart fibroblasts. These findings have been observed: in normal culture medium the closest approach of the cell surface to substrate in its adhesions is ca. 100 A, much of the cell surface lying farther away; chemical treatments which bring the cell surface to near its charge reversal point reduce the closest approach of adhesions to <50 A, probably to <30 A; chemical treatments which increase surface charge increase the nearest approach of cell and substrate in adhesions from ca. 100 A; high osmotic concentration of a non-polar substance, i.e. sucrose, does not affect the distance between cell and substrate in the adhesions. In addition, optical evidence indicates that there is no extracellular material between cell and glass in the adhesions. When cells de-adhere from glass, they appear not to leave fragments behind. The adhesive sites in these fibroblasts appear to be confined to the edge of the side of the cell facing the substrate and to the pseudopods. The significance of this is discussed in relation to the phenomenon of contact inhibition. Evidence is presented that the mechanism of cell adhesion does not involve calcium atoms binding cells to substrate by combining with carboxyl groups on cell surface, substrate, and with a cement substance. Osmium tetroxide fixation results in a final separation of 100 to 200 A between cell and substrate: there are reasons for thinking that this fairly close approach to the condition in life is produced as an artefact. The results can be accounted for only in terms of the action of electrostatic repulsive forces and an attractive force, probably the van der Waals-London forces. Biological arguments suggest that these results are equally applicable for cell-to-cell adhesions.

Animals↗

PubMed1997

Quantitation of cell-matrix adhesion using confocal image analysis of focal contact associated proteins and interference reflection microscopy.

We have developed an approach for the quantitation of vinculin, a focal contact associated protein, based on a multimodal confocal microscopy and image analysis. Vinculin spot distribution was imaged in confocal fluorescence microscopy and the corresponding focal contacts were imaged in confocal interference reflection microscopy. These images were analyzed with a SAMBA image cytometer. The image analysis program provided 12 morphometric features describing cellular area, shape, and proportions of vinculin spots as well as six topographical features describing the distribution of vinculin and the relative overlap of vinculin and focal contacts. This approach was applied to the study of rat osteosarcoma cells submitted to mechanical stresses: successions of 2g and 0g accelerations during a series of parabolic flights. The measured features were assessed by means of correlation analysis and stepwise discriminant analysis. After correlation analysis, only ten parameters were retained. Quantitation of cell morphological parameters indicated that cell area was significantly affected by gravitational stresses as well as vinculin distribution. Cell area was reduced by 50% and vinculin spots were restricted to cell periphery. Cell adhesion measured by IRM decreased significantly in the first part of the flight and remained stable at the end of the flight. These results suggest that cell-matrix adhesion is affected by gravitational stresses. Image analysis provides useful tools to investigate focal adhesion re-organization under different physiological stimuli.

Animals↗