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At least 109 records · Page 6Linked to original sources

Platelet adherence and detachment with adsorbed fibrinogen: a flow study with a series of hydroxyethyl methacrylate-ethyl methacrylate copolymers using video microscopy.

The adhesion and detachment of platelets were studied on glass coatings of a series of copolymers of hydroxyethyl methacrylate (HEMA) and ethyl methacrylate (EMA) with preadsorbed fibrinogen. Observations of the interactions of acridine-orange-labeled washed platelets with these surfaces from a flowing (500 s-1 wall shear rate) suspension in Tyrode's solution containing albumin and red cells were made with epifluorescent video microscopy (EVM). In some cases preadsorbed materials were incubated for 24 h, during which little or no loss of protein occurred. Protein surface concentration, by itself, was a poor indicator of expected cell adhesion and morphology. Surface chemistry was a second important factor which must be considered. A third observation is that for the 100% EMA copolymer, 24 h of incubation led to a large reduction in platelet adhesion when compared to the 100% EMA material without incubation. For the 0% and 100% EMA polymers, the percentage of contacting platelets which adhere and detach is greater for the 24-h incubation cases than for those not incubated. These results led to the conclusion that our most hydrophilic surface favors adhesion with detachment, transient cell contact, over long-term adhesion, as does incubation of adsorbed protein. A brief discussion is presented of a possible connection between this behavior and platelet consumption in vivo for hydrogels.

Adsorption↗

Intracellular oxygen measurements of mouse liver cells using quantitative fluorescence video microscopy.

Our currently developed fluorescence video microscope can measure fluorescence intensities with an error of +/- 1.5% of full scale in 65536 different positions of a microscope field. With a video frame freeze acquisition time of 33 ms, time-dependent changes of this order of time or slower can be followed. Using cells which have absorbed pyrene-1-butyrate to an intracellular concentration of 0.05 to 1 mM, the changes in fluorescence intensity with oxygen concentration are easily measured. The spatial resolution for data collection is 0.5 micron when a 54X objective is used. The individual Stern-Volmer quenching constants of each individual pixel were measured for agar slices and mouse liver cells treated with pyrenebutyric acid. The distribution of quenching constants for agar follows a normal curve about a mean value of 16 . 10(-4) torr-1. The data for mouse liver cells gave a non-normal distribution of quenching constants with a mean value of 18 . 10(-4) torr-1. The greater spread of the data from cells is interpreted as evidence for a real biological variation in the solubility coefficient of oxygen in different locations within the cell. In all the cells examined, this distribution has been observed to be non-random and appears to be associated with specific cell structures.

Animals↗

Video microscopy of intracellular pH in primary cultures of rabbit proximal and early distal tubules.

The purpose of this study was to investigate intracytoplasmic pH (pHi) regulation in primary cultures of proximal (PCT) and distal bright (DCTb) convoluted tubules. PCT and DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The cultured epithelia were grown on semi-transparent permeable supports. The pHi was determined by video microscopy and digital image processing using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and measuring the ratio of BCECF fluorescence excited by two successive wavelengths (490 nm and 450 nm). Resting pHi values, determined in bicarbonate-free medium (extracellular pH: 7.40), were 7.25 +/- 0.02 (n = 23) and 7.17 +/- 0.04 (n = 30) for cultured PCT and DCTb respectively. After the acid-loading procedure, cultured proximal cells recovered their pHi by means of the classic Na+/H+ antiporter, sensitive to amiloride and located in the apical membrane only. In cultured DCTb part of the pHi recovery was mediated by a Na+/H+ exchange present in the basolateral side. Moreover, at physiological initial pHi values, chloride removal from the apical solution caused the pHi to increase in the presence of bicarbonate. In acidified cultured DCTb cells, a partial pHi recovery was induced in sodium-free media by 15 mM HCO(-3) in the presence of an outward chloride gradient. This pHi change was completely abolished by 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid (1 mM). These data suggest that DCTb cells possess in apical anion/base exchanger that resembles the Na(+)-independent Cl-/HCO(-3) exchanger.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

In vitro evaluation of platelet/biomaterial interactions in an epifluorescent video microscopy combined with a parallel plate flow cell.

Suitable evaluation systems are critical for ranking various biomaterials in order to develop a method to design and synthesize nonthrombogenic biomaterials. We have recently developed an in vitro test system to evaluate platelet/biomaterial interactions in whole blood. The system consists of a parallel plate flow cell and epifluorescent video microscopy (EVM). A glass coverslip coated with a polymer was incorporated into the flow cell, and blood was perfused using a syringe pump via a polymer-coated PVC tubing connected to the flow cell. Whole human blood was anticoagulated with heparin (2 U/ml), and the platelets were labeled with the fluorescent dye mepacrine (5 microM). This system permitted real-time and dynamic observations of platelet/biomaterial interactions in whole blood under a defined flow condition. In order to evaluate the feasibility of this system, two different segmented polyether-polyurethanes (SPEUs), PU-PTMG(650) and PU-PTMG(2000), were chosen as test polymers. Surface characteristics verified with electron spectroscopy for chemical analysis (ESCA) and contact angle measurements showed similar results in both SPEUs. Blood was perfused at a wall shear rate of 200 s-1 for 20 min. Excitation light was applied for 2 s at 1 min intervals. The real-time image was then analyzed at each time point for the percentage of surface area of platelet coverage. Plasma beta-thromboglobulin (beta-TG) levels were also measured before and after each run. PU-PTMG(650) showed a significantly higher number of adhered platelets than PU-PTMG(2000) at each time point.(ABSTRACT TRUNCATED AT 250 WORDS)

Biocompatible Materials↗

Dynamic behaviors of growth cones extending in the corpus callosum of living cortical brain slices observed with video microscopy.

During development, axons of the mammalian corpus callosum must navigate across the midline to establish connections with corresponding targets in the contralateral cerebral cortex. To gain insight into how growth cones of callosal axons respond to putative guidance cues along this CNS pathway, we have used time-lapse video microscopy to observe dynamic behaviors of individual callosal growth cones extending in living brain slices from neonatal hamster sensorimotor cortex. Crystals of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) were inserted into the cortex in vivo to label small populations of callosal axons and their growth cones. Subsequently, 400 microns brain slices that included the injection site, the corpus callosum, and the target cortex were placed in culture and viewed under low-light-level conditions with a silicon-intensified target (SIT) camera. Time-lapse video observations revealed striking differences in growth cone behaviors in different regions of the callosal pathway. In the tract, which is defined as the region of the callosal pathway from the injection site to the corresponding target cortex, growth cones advanced rapidly, displaying continual lamellipodial shape changes and filopodial exploration. Forward advance was sometimes interrupted by brief pauses or retraction. Growth cones in the target cortex had almost uniform compact shapes that were consistently smaller than those in the tract. In cortex, axons adhered to straight radial trajectories and their growth cones extended at only half the speed of those in the tract. Growth cones in subtarget regions of the callosum beneath cortical targets displayed complex behaviors characterized by long pauses, extension of transitory branches, and repeated cycles of collapse, withdrawal, and resurgence. Video observations suggested that extension of axons into cortical targets could occur by interstitial branching from callosal axons rather than by turning behaviors of the primary growth cones. These results suggest the existence of guidance cues distinct for each of these callosal regions that elicit characteristic growth cone behaviors.

Animals↗

Elastic properties of 2D colloidal crystals from video microscopy.

Elastic constants of two-dimensional (2D) colloidal crystals are determined by measuring strain fluctuations induced by Brownian motion of particles. Paramagnetic colloids confined to an air-water interface of a pendant drop are crystallized under the action of a magnetic field, which is applied perpendicular to the 2D layer. Using video microscopy and digital image processing we measure fluctuations of the microscopic strain obtained from random displacements of the colloidal particles from their mean (reference) positions. From these we calculate system-size dependent elastic constants, which are extrapolated using finite-size scaling to obtain their values in the thermodynamic limit. The data are found to agree rather well with zero-temperature calculations.

Journal Article↗

Compartmentalization of algal bioluminescence: autofluorescence of bioluminescent particles in the dinoflagellate Gonyaulax as studied with image-intensified video microscopy and flow cytometry.

Compartmentalization of specialized functions to discrete locales is a fundamental theme of eucaryotic organization in cells. We report here that bioluminescence of the dinoflagellate alga Gonyaulax originates in vivo from discrete subcellular loci that are intrinsically fluorescent. We demonstrate this localization by comparing the loci of fluorescence and bioluminescence as visualized by image-intensified video microscopy. These fluorescent particles appeared to be the same as the previously described in vitro "scintillons." We attribute the endogenous fluorescence to that of the bioluminescence substrate, luciferin, because (a) the fluorescence excitation and emission characteristics are comparable, (b) the autofluorescence is lost after exhaustive stimulation of bioluminescence, and (c) the fluorescence of discharged particles in vitro can be restored by adding luciferin. The fluorescence in vivo exhibits a standard property of circadian (daily) rhythmicity: under constant environmental conditions, the intensity of the particle fluorescence fluctuates cyclically (it is maximal during the night phase and is low during the day). Thus, luciferin is localized within the cell at discrete loci from which the bioluminescence emanates; the cellular quantity of luciferin is rhythmically modulated by the circadian clock.

Dinoflagellida↗

Platelet adherence and detachment: a flow study with a series of hydroxyethyl methacrylate-ethyl methacrylate copolymers using video microscopy.

The adhesion and detachment of platelets were studied on glass coatings of a series of copolymers of hydroxyethyl methacrylate (HEMA) and ethyl methacrylate (EMA). Observations of the interactions of mepacrine labelled washed platelets with these surfaces from a flowing (500 s-1 wall shear rate) suspension in Tyrode's solution containing albumin and red cells were made with epifluorescent video microscopy (EVM). Total platelet adhesion, including platelets which adhere on first contact and platelets which attach temporarily before adhesion, and the number of detaching platelets were minimal for the 0 and 20% EMA copolymers, reached a maximum for the 50% EMA copolymer and showed reduced values for the 80% and 100% EMA copolymers. For the 50, 80, and 100% EMA copolymers, the adhesion values expressed, as a percentage of total contacting platelets, were not different. Albumin adsorption to these copolymers shows a continuous increase from the 0% to the 100% EMA copolymer. It is likely that the peak in platelet adhesion at the 50% EMA composition is related to: low protein adsorption on the 0 and 20% EMA copolymers, too little albumin adsorption to block adhesion on the 50% EMA copolymer, and full-scale blocking on the 80 and 100% EMA copolymers due to greater albumin adsorption.

Adult↗

Video-microscopy for analysis of molecular dynamics in cells.

Real-time analysis of molecular dynamics in living cells was studied by developed video-microscopes. Two new detective methods were reported, one is for analysis of ciliary movement and the other is the qualitative analysis of exocytosis of insulin-containing granules with a video-enhanced light/fluorescent microscope. For analysis of ciliary movement, glass beads were migrated in the flow. The migration speed parallel to the flow produced by ciliary beating was used as an index of the beating activity. When tracheal epithelium isolated from mouse was incubated with ambroxol, and expectorant known to activate ciliary beat frequency, the floating speeds of glass beads were changed with 1 min of incubation. The results suggest that the present method is useful not only for screening of expectorants but also for the study of molecular mechanisms underlying ciliary beat of tracheal epithelium. Visualization of the moment of the release of contents from insulin-containing granules was achieved using video-enhanced fluorescent microscopy in MIN6 cells of mouse insulinoma cell line. A fluorescent amino acridine dye, quinacrine, was found to be incorporated into low-pH secretory granules, including insulin, in the cells. The granules which incorporated quinacrine emitted a slightly blue-green fluorescence. Upon stimulation with glucose, release of the quinacrine fluorescence from granules were observed. The present method would be useful for quantitative analysis of secretion of insulin from MIN6 cells as well as pancreatic beta-cells.

Analysis of Variance↗

How to evaluate the quality in a system of video-microscopy in the field of anatomic pathology.

The acquisition of a video microscope image is explained by the concept of modulation transfer function to describe the quality of a microscope which should target a sensor of a video camera to allow around 50 pairs of lines per mm with a good contrast in the video-monitor. Taking this into consideration, high aperture lenses, a microscope with infinity optics, a low power video adapter and a high quality camera must be used.

Humans↗

Multiparameter digitized video microscopy of toxic and hypoxic injury in single cells.

There is no clear picture of the critical events that lead to the transition from reversible to irreversible injury. Many studies have suggested that a rise in cytosolic free Ca2+ initiates plasma membrane bleb formation and a sequence of events that lead ultimately to cell death. In recent studies, we have measured changes in cytosolic free Ca2+, mitochondrial membrane potential, cytosolic pH, and cell surface blebbing in relation to the onset of irreversible injury and cell death following anoxic and toxic injury to single hepatocytes by using multiparameter digitized video microscopy (MDVM). MDVM is an emerging new technology that permits single living cells to be labeled with multiple probes whose fluorescence is responsive to specific cellular parameters of interest. Fluorescence images specific for each probe are collected over time, digitized, and stored. Image analysis and processing then permits quantitation of the spatial distribution of the various parameters with the single living cells. Our results indicate the following: The formation of plasma membrane blebs accompanies all types of injury in hepatocytes. Cell death is a rapid event initiated by rupture of a plasma membrane bleb, and it is coincident with the onset of irreversible injury. An increase of cytosolic free Ca2+ is not the stimulus for bleb formation or the final common pathway leading to cell death. A decrease of mitochondrial membrane potential precedes the loss of cell viability. Cytosolic pH falls by more than 1 pH unit during chemical hypoxia. This acidosis protects against the onset of cell death.

Animals↗

Flexible method to obtain high sensitivity, low-cost CCD cameras for video microscopy.

A simple method is described to extend image exposure times in video-rate CCD cameras and thereby, increase their sensitivity and reduce noise level of low-light images. Most commercial video cameras lack the capability of extending image exposures since they operate regular television timing formats. The technique described here implements the control of the exposure times by selectively gating the image readout from the CCD sensor. This prevents the cyclic clearing of photo-charges occurring at regular video-rates, allowing image integration beyond the duration of single video field periods. Image readout is controlled by the duration of external gating pulses, giving the camera an efficient operational versatility under different light conditions. This technique is applicable to standard monochrome and color CCD cameras. The evaluations described here using this technique show that the light sensitivity of an standard video-rate CCD camera can be significantly improved, generating high quality images at low-light levels. These were comparable to those obtained with image intensifiers or intensified video cameras. Cameras are still compatible with regular video equipment, since this technique preserves the normal TV synchronization signals. Results in simulated and real experimental situations confirmed that this technique enables the use of affordable video-rate CCD cameras for a variety of fluorescence microscopy and optical recording applications.

Animals↗

Flow electrification in nonaqueous colloidal suspensions, studied with video microscopy.

Flow electrification in nonaqueous suspensions has been scarcely reported in the literature but can significantly affect colloidal stability and (phase) behavior, perhaps even without being recognized. We have observed it in shear flow experiments on concentrated binary suspensions of hydrophobized silica particles in chloroform. In this low-polarity solvent, electrical charges on the large-particles' surfaces manifest themselves via long-ranged forces, because hardly any screening can take place through counterions. By shearing the suspension for a prolonged time, we could demonstrate that the effective interactions between the large particles change from weakly attractive (due to the small particles) to strongly repulsive (due to acquired Coulomb interactions). One of the conditions required for flow electrification was the presence of a glass surface in the shear cell. A spectacular manifestation of the phenomenon was observed with confocal video microscopy. First, the formation of large-particle aggregates was seen, while subsequently (over a long shearing time) the aggregates disintegrated into small entities, mostly primary particles. The spatial distribution of these entities in the quiescent state after stopping the flow showed evidence for acquired long-range repulsion. The occurrence of flow electrification was further corroborated by control experiments, where no flow was imposed, antistatic agent was added, or the glass bottom was coated with a conducting (indium tin oxide, ITO) layer: here, the aggregates kept growing until they became very large. To further diagnose the phenomenon, we have also done experiments in which an external electric field was applied (via the ITO layer) to an aggregated suspension. When the lower electrode was given the lowest potential, the aggregates were found to move away from the bottom and disintegrate. The qualitative similarity hereof with the flow electrification experiment suggests that in the latter, the glass acquired negative charges. After prolonged application of an external electric field, we observed segregation into regions enriched in large particles and regions completely depleted of them. In the quiescent fluid these regions exist as isolated units, but in shear flow they merge into bands, a behavior which resembles shear banding.

Journal Article↗

A quantitative method for the detection and localization of quantum-limited events from radionuclides in cells and tissue sections by computer-enhanced video microscopy.

Cellular dynamics often involve extremely low concentrations of biologically active substances, which can be radiolabeled and detected, localized and quantitated by autoradiography. The latter may require exposures from a few days to many months. The objective of this research was to demonstrate the feasibility of reducing this long period of data collection by one to two orders of magnitude, while maintaining or improving the spatial resolution and localization in tissues and the quantitative characteristics inherent in autoradiography. A mathematical model describing the complete system was generated using energy partition calculations to estimate photon production via scintillant per H3 beta particle emission and to estimate the subsequent photon capture based upon imaging system parameters and microscope geometry. Calculations showed that, typically, a single tritium beta particle produces a maximum of 5.8 X 10(3) photons. A photon-limited camera and microscope imaging system were selected and optimized in conjunction with a specially developed physical scintillation model. Results showed that the number of detected photoevents increases monotonically with both signal integration time and, independently, with the concentration of the radionuclide. Consequently, this work demonstrates that video microscopy imaging methods can spatially and temporally quantify very low concentrations of radiolabeled substances and can reduce data acquisition times.

Beta Particles↗

Extension of long cellular processes of hepatic stellate cells cultured on extracellular type I collagen gel by microtubule assembly: observation utilizing time-lapse video-microscopy.

Hepatic stellate cells cultured on or in freshly prepared type I collagen gel as a substratum were induced to elongate long cellular processes. The extension of the cellular processes was monitored by using video-enhanced optical microscopy. The cellular processes seemed to extend along the extracellular type I collagen fibers. Once extended cellular processes after overnight culture on type I collagen gel were retracted by cytoskeleton degradation with colchicine or cytochalasin B. The cellular processes were also retracted by treatment with protein kinase inhibitor, herbimycin A or staurosporin, or with phosphatidylinositol 3-kinase inhibitor, wortmannin. The effects of colchicine, herbimycin A, staurosporin, or wortmannin were drastic, and the cells were finally changed to a round shape within a few hours, as seen also after cold-treatment at 4 degrees C. Cytochalasin B also time-dependently retracted the extended cellular processes. These results indicated that the cultured stellate cells were induced to elongate cellular processes by cell surface binding to type I collagen fibrils, followed by protein or phosphatidylinositol phosphorylation and finally F-actin and microtubule assembly. Extended long cellular processes seem to reflect the in vivo structure of hepatic stellate cells, and molecular mechanism for the extension and maintenance of cellular processes was proposed.

Animals↗

VIPER: a general-purpose digital image-processing system applied to video microscopy.

This paper describes VIPER, the video image-processing system Erlangen. It consists of a general purpose microcomputer, commercially available image-processing hardware modules connected directly to the computer, video input/output-modules such as a TV camera, video recorders and monitors, and a software package. The modular structure and the capabilities of this system are explained. The software is user-friendly, menu-driven and performs image acquisition, transfers, greyscale processing, arithmetics, logical operations, filtering display, colour assignment, graphics, and a couple of management functions. More than 100 image-processing functions are implemented. They are available either by typing a key or by a simple call to the function-subroutine library in application programs. Examples are supplied in the area of biomedical research, e.g. in in-vivo microscopy.

Animals↗

Phagosome formation in Paramecium: roles of somatic and oral cilia and of solid particles as revealed by video microscopy.

The roles of somatic and oral cilia and solid particles during digestive vacuole (DV) formation in Paramecium multimicronucleatum were investigated using video-enhanced and immunofluorescence microscopy. Membrane incorporation into DVs was found to increase linearly with increasing particle concentration. The rate of discoidal vesicle transport to the cytopharynx was not affected by particles, showing that particles are not required for membrane trafficking to the cytopharynx. However, the presence of particles leads to an increased membrane fusion between the cytopharyngeal membrane and the discoidal vesicles. When live cells lost their somatic cilia on the left-ventral side anterior to the oral region due to deciliation, membrane incorporation into newly formed DVs was strongly inhibited. Using video-enhanced microscopy, latex beads were seen to be loaded along the quadrulus on the dorsal surface of the buccal cavity, but few beads were seen next to the dorsal and ventral peniculi. Particle sequestration into a pre-formed nascent digestive vacuole (NDV) was studied in Triton X-100-permeabilized cells whose ciliary beating was reactivated by the addition of Mg-ATP. Both beat frequency and the percentage of cells containing bead-labeled NDV were dependent on the Mg-ATP concentration: the higher the beat frequency, the higher the percentage of cells with a bead-labeled NDV. These results suggest that ciliary beating is probably the only mechanism required for particle accumulation in the NDV, while a coordinated beating of the somatic cilia on the left-ventral side anterior to the oral region as well as the quadrulus moves particles into the NDV. The beating of the peniculi may somehow prevent the backward flow of particles out of the NDV.

Animals↗

Image processing for combined bright-field and reflection interference contrast video microscopy.

Image processing algorithms for automatic extraction of cell body contours and cell-substratum contacts from video images, which were obtained by bright-field microscopy and reflection interference contrast microscopy, respectively, are described. Double-view imaging, which combines these two optical techniques, is used to investigate the relationship between cell-to-substratum adhesion and cell shape changes during locomotion of the amoeboid cells of Dictyostelium discoideum. Contact areas of cells are extracted from reflection interference contrast images via a routine which performs binarisation on the basis of threshold estimation, as it is calculated by a histogram minimum method. Boundaries of cells are extracted from bright-field images by utilising and algorithm that includes background subtraction and binarisation based on texture discrimination by means of a rank operator.

Animals↗