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PubMed2006

Analysis of DNA recombination and repair proteins in living cells by photobleaching microscopy.

DNA double strand break repair through homologous recombination has been shown biochemically to require the coordinated action of the RAD52 group of proteins, including the DNA strand exchange protein Rad51. We have started to develop experimental tools to investigate the close cooperation of homologous recombination proteins in living cells, where proteins operate in the context of chromatin and in the presence of other nuclear processes. This chapter describes in detail methods to establish cell lines stably expressing green fluorescent protein -tagged recombination proteins and photobleaching techniques to investigate the behavior of the proteins with the use of live cell video microscopy. Fluorescence recovery after photobleaching (FRAP), fluorescence loss after photobleaching (FLIP), and their combination in the same cell are useful techniques to gain insights into the dynamic behavior of the recombination proteins. Parameters such as diffusion rates and mobile versus immobile fractions before and after DNA damage induction can be obtained. In addition, residence times of recombination proteins at sites of DNA damage can be determined. Through the application of FRAP and FLIP it is possible to establish whether proteins are present in the same multiprotein complex, whether this is affected by DNA damage induction, and whether proteins dynamically associate with and dissociate from sites of DNA damage.

Animals↗
PubMed1989

The circulation of the human limbus.

The superficial circulations of the anterior segment of the human eye have been studied by the following techniques: photographic and video low-dose fluorescein angiography, video-microscopy using red-free light and stereoscopic colour photography. The results are combined to give a dynamic description of the limbal circulation, including its arterial supply and venous drainage.

Anterior Eye Segment↗
PubMed1992

Effects of lung inflation and blood flow on capillary transit time in isolated rabbit lungs.

In a previous study, direct measurements of pulmonary capillary transit time by fluorescence video microscopy in anesthetized rabbits showed that chest inflation increased capillary transit time and decreased cardiac output. In isolated perfused rabbit lungs we measured the effect of lung volume, left atrial pressure (Pla), and blood flow on capillary transit time. At constant blood flow and constant transpulmonary pressure, a bolus of fluorescent dye was injected into the pulmonary artery and the passage of the dye through the subpleural microcirculation was recorded via the video microscope on videotape. During playback of the video signals, the light emitted from an arteriole and adjacent venule was measured using a video photoanalyzer. Capillary transit time was the difference between the mean time values of the arteriolar and venular dye dilution curves. We measured capillary transit time in three groups of lungs. In group 1, with airway pressure (Paw) at 5 cmH2O, transit time was measured at blood flow of approximately 80, approximately 40, and approximately 20 ml.min-1.kg-1. At each blood flow level, Pla was varied from 0 (Pla less than Paw, zone 2) to 11 cmH2O (Pla greater than Paw, zone 3). In group 2, at constant Paw of 15 cmH2O, Pla was varied from 0 (zone 2) to 22 cmH2O (zone 3) at the same three blood flow levels. In group 3, at each of the three blood flow levels, Paw was varied from 5 to 15 cmH2O while Pla was maintained at 0 cmH2O (zone 2).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Calcium dynamics in thorny excrescences of CA3 pyramidal neurons.

Confocal laser scanning microscopy was used to visualize Ca2+ transients in a particular type of dendritic spine, known as a thorny excrescence, in hippocampal CA3 pyramidal neurons. These large excrescences or thorns, which serve as the postsynaptic target for the mossy-fiber synaptic inputs, were identified on the basis of their location, frequency, and size. Whole cell recordings were made from superficial CA3 pyramidal neurons in thick hippocampal slices with the use of infrared video microscopy; cells with proximal apical dendrites close to the surface of the slice were selected. Changes in intracellular Ca2+ levels were monitored by imaging changes in fluorescence of the dyes Calcium Green-1 and Fluo-3. Dual-emission fluorescence imaging was also employed with the use of a combination of Fluo-3 and the Ca2+-insensitive dye seminaphthorhodafluor-1. This method was used to decrease the potential influence of background fluorescence on the calculated changes in intracellular Ca2+ concentration ([Ca2+]i). Somatic depolarization produced increases in [Ca2+]i in both the thorn and the immediately adjacent dendrite. Changes in [Ca2+]i were time locked with the onset of depolarization and the decay began immediately after the termination of depolarization. The peak increase in the Ca2+ signal was significantly greater in the thorns than in the adjacent dendritic shafts. With the use of high-temporal-resolution methods (line scans), differences were also seen in the time course of Ca2+ signals in these two regions. The decay time constants of the Ca2+ signal were faster in thorns than in the adjacent dendritic shafts. These observations suggest that voltage-gated Ca2+ channels are localized directly on the dendritic spines receiving mossy-fiber input. Furthermore, Ca2+ homeostasis within thorny excrescences is distinct from Ca2+ regulation in the dendritic shaft, at least over brief time periods, a finding that could have important implications for synaptic plasticity and signaling.

Animals↗
PubMed1986

Electropore diameters, lifetimes, numbers, and locations in individual erythrocyte ghosts.

Low light level video microscopy was used to study the diameter, lifetime, number, and location characteristics of electric field-induced pores (electropores) in erythrocyte ghosts. The diameter of electropores was probed by following the efflux of soluble fluorescent-tagged molecules out of the resealed ghost cytoplasmic compartments. After reaching a peak radius of at least 8.4 nm the electropores resealed within 200 ms to a radius of about 0.5 nm and stayed at that radius thereafter. Video sequences clearly show that pores are induced preferentially in the cathodal hemisphere. Pores induced in the hemisphere facing the positive electrode were either never greater than 0.5 nm in radius, much smaller in number if they were greater than 0.5 nm in radius, or shorter lived. Calculations indicated that an upper limit of 700 electropores were induced per membrane.

Cell Membrane Permeability↗
PubMed1989

Interactive computer-video modules for health sciences education.

Advances in electronic image recording and computer technology have resulted in a remarkable increase in the power and flexibility of interactive computer-video teaching systems. The University of Washington Health Science Videodisc Development Group first demonstrated a laser videodisc controlled by a remote central computer in 1980. Even this rudimentary unit highlighted basic medical informatics principles including: rapid accessibility; a "generic" or multi-purposed format; ease of computer control; and large collections of valid, rigorously reviewed images. Advances in medical informatics have led to the development of the following previously undescribed series of teaching units: 1. The hypertext programs Hypercard, Linkway, and Guide have been used with videodiscs to develop easy-to-use instructional and reference materials. These materials demonstrate the ease with which a computer-naive instructor may develop new programs and the advantages that the intuitive nature of these programs brings to student users. 2. Patient simulations using single and double screens plus pre-defined knowledge structures; 3. Interactive single topic tutorials using preset knowledge structures; 4. A key-word-based disc searching system; 5. Electronic video microscopy; 6. A series of programs developed independently by health science faculty who have purchased multi-purpose videodiscs that demonstrate the flexibility of the multi-purpose or "generic": collection concept.

Computer Simulation↗
PubMed2002

Process of apoptosis induced by TNF-alpha in murine fibroblast Ltk-cells: continuous observation with video enhanced contrast microscopy.

Apoptosis is originally defined by unique morphological changes of dying cells, and the biochemical hallmark associated with apoptosis is internucleosomal DNA fragmentation. However, few report has shown the precise time course of the apoptotic events. The present study was designed to try to clarify apoptotic processes using a video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy. The morphological changes of murine fibroblast Ltk-cells treated with TNF-alpha were divided into four stages: (i) pre-apoptotic, (ii) cytoplasmic shrinkage, (iii) membrane blebbing, and (iv) ballooning. Almost of the cells underwent cytoplasmic shrinkage and membrane blebbing within 6 hours after TNF-alpha exposure, and at about 9 hours, they were in the ballooning stage. Based on these data, we investigated the relationship between morphological changes and other biochemical features. The earliest event was exposure of phosphatidyl-serine at the cytoplasmic membrane, which was already observed in the pre-apoptotic stage. Loss of mitochondrial membrane potential was observed in the cytoplasmic shrinkage stage. Caspase-8/-3 activities already started increasing in the pre-apoptotic stage, and reached their peak at 6 hours after TNF-alpha exposure. DNA fragmentation occurred in the late phase of the membrane blebbing.

Animals↗
PubMed1991

Leukocyte adhesion and cell death following orthotopic liver transplantation in the rat.

The purpose of these experiments was to employ video microscopy to identify early pathological changes after orthotopic liver transplantation in the rat. Liver transplantation was performed using the cuff technique. Survival was greater than 90% when livers were stored for 1 hr in Ringer's solution prior to transplantation (survival conditions), whereas all rats died following storage of grafts for 4 hr in cold Euro-Collins solution (nonsurvival conditions). Postoperatively, each recipient animal was anesthetized, the abdomen was opened, and the liver was placed on the stage of an inverted fluorescence microscope equipped with a low-light ISIT video camera. Precautions were taken to prevent the liver surface from drying. The fluorescent dyes fluorescein sodium (1.0 mumol/kg), acridine orange (2.5 mumol/kg), and propidium iodide (1 mumol/kg) were injected intravenously to label hepatocytes, polymorphonuclear leukocytes, and the nuclei of irreversibly damaged cells, respectively. In livers from untransplanted rats, movement of labeled leukocytes through the hepatic sinusoids was smooth and rapid (velocity, 500-550 microns/sec) and margination (adhesion) of cells was minimal (less than 1%). After transplantation, however, velocity was diminished 2-3-fold, and margination was increased to 10% of leukocytes in survival groups and to 40% in nonsurvival groups 4 hr postoperatively. In the nonsurvival groups, irreversible cell death detected by propidium iodide fluorescence was minimal 15 min after transplantation--however, massive cell damage was detected 4 hr postoperatively. In addition, serum transaminases and necrosis were higher at 4 hr than 15 min postoperatively. Taken together, these data demonstrate that leukocyte margination increases following orthotopic liver transplantation and is followed rapidly by cell death. These events likely play a role in the mechanism of early graft failure following transplantation.

Animals↗
PubMed1987

A model for chronic study of vascular morphology and reactivity of physically intact rat skin microcirculation.

A non-invasive, non-traumatic model has been developed for long term study of the vascular anatomy, blood cell velocity and vascular reactivity of the cutaneous microcirculation of the rat. In the anesthetized rat, the hairless area of a toe close to the nail could be visualized by intravital video microscopy without manipulation of the skin. Good image quality of the vascular bed was obtained with the use of an incident illuminator equipped with a 50% mirror at 45 degrees to the optical axis and a polarizer-analyzer system in combination with a mercury lamp. This microscope system allows for visualization of the superficial vasculature of the skin, while microvessels can be repeatedly identified for several weeks. To test for vascular reactivity off-line analysis of blood cell velocity was performed in the control situation and during reactive hyperemia, using a video dual-window system and a cross-correlator. The mean control velocity in venules selected for testing of vascular reactivity was 70 micron/s. In these vessels a reproducible pattern of reactive hyperemia was seen with a mean peak velocity of 655 micron/s following arterial occlusion of 180 s. Possible applications of the model are discussed.

Animals↗
PubMed1992

A cell division counter exists in the chick embryo myogenic lineage.

A computer assisted timelapse video microscopy system was developed to study the behaviour of individual chick embryo myogenic cells in culture. With this system it is possible to monitor and photograph the development of 50-100 myogenic clones simultaneously. Photos were made with a frame grabber and image compressor and were stored in digital form on the computer's hard disc. Colonies derived from 10-day chick embryo myogenic were followed over a period of 3 days. The cultures were then fixed, reacted first with a mouse monoclonal antibody against sarcomeric myosin, then with peroxidase-labelled goat anti-mouse IgG, and stained with diaminobenzidine to detect antibody binding. The dishes were then replaced into the video system and the computer was used to find the originally photographed colonies. Cells which bound the antibody against sarcomeric myosin were identified in the last image stored for each colony. The computer was then used to retrieve the images from each colony in reverse order and to determine the lineage ancestry of every antibody-positive cell in each clone. Several hundred colonies and many hundreds of cell divisions have been examined thus far. In all but one clone, every antibody-positive cell was produced by a symmetrical cell division in which its sister was also antibody-positive (or died). Antibody-positive cells were generated by a sequence of no more than 4 symmetrical cell divisions. Thus, most colonies consisted of 1, 2, 4, 8 or 16 nuclei in terminally differentiated muscle cells. Those colonies in which cell numbers was not a power of 2 were always produced by cell death or detachment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Platelet-activating factor induces pseudopod formation in calcitonin-treated rabbit osteoclasts.

We demonstrated previously that platelet-activating factor (PAF), a potent inflammatory mediator, acts on osteoclasts to elevate cytosolic [Ca2+] and stimulate resorption. However, it is not clear whether the effects of PAF on resorptive activity are direct or indirect. In the present study, we investigated the effects of PAF on osteoclast motility. Osteoclasts were isolated from the long bones of neonatal rabbits, and cell motility and morphology were monitored using time-lapse video microscopy. Calcitonin, a hormone known to induce retraction of pseudopods and inhibit resorptive activity, was used to render osteoclasts quiescent. Within 10 minutes of calcitonin treatment (100 ng/ml, final), pronounced retraction of pseudopods was observed in 68 of 112 cells tested. When PAF (200 nM, final) was added 10 minutes after calcitonin treatment, pseudopods were evident 1 h later in 15 of 37 calcitonin-responsive cells tested. In contrast, pseudopods were evident in only 4 of 31 calcitonin-responsive cells treated with control solutions (PAF-vehicle or S-PAF, the biologically inactive stereoisomer of PAF). Pseudopod formation was quantified by measuring the planar area of pseudopods with a computer-based video analysis system. When assessed 60 minutes following PAF treatment, the pseudopod area was significantly greater in PAF-treated cells than in control cells. In some calcitonin-treated osteoclasts, PAF induced pseudopod formation when applied focally using an extracellular micropipette, consistent with a direct action of PAF. We conclude that PAF directly induces pseudopod formation in calcitonin-inhibited osteoclasts, a morphologic response indicative of osteoclast activation.

Animals↗
PubMed2004

Exploring molecular motors and switches at the single-molecule level.

Single-molecule techniques have propelled an impressive number of biophysical studies during the last decade. From relatively simple video-microscopy techniques, to sophisticated manipulation and detection apparata, single-molecule techniques are capable of tracking the movements and the reaction trajectories of single enzymatic units. By observing microspheres attached to biomolecules it is possible to follow the motion of molecular motors, or to detect conformational "switching" induced by regulatory proteins. Micromanipulation tools like optical tweezers have been widely applied to understand the mechanisms of linear molecular motors, and have allowed the measurement of the elementary steps and the forces produced by several motor proteins, including myosin, kinesin, and dynein. New experimental assays based on magnetic or optical "wrenches," which are able to apply and detect torques on rotary motors and biopolymers, are opening new possibilities in this field. Here, established and emerging magneto-optical manipulation and video-tracking techniques are reviewed, in the perspective of single molecular motors and regulatory proteins studies.

Adenosine Triphosphate↗
PubMed2004

Importance of platelets and fibrinogen in neutrophil-endothelial cell interactions in septic shock.

OBJECTIVE: To examine the role of platelets, fibrin, and adhesion molecules in mediating neutrophil-endothelial cell interactions in septic shock. DESIGN: Controlled experiments using phase contrast microscopy to examine neutrophil, platelet, and endothelial cell interactions in flowing cell suspensions under simulated physiologic conditions. SETTING: University research laboratory. PATIENTS: Adult patients with septic shock and normal volunteers. INTERVENTIONS: Microslides were coated with human umbilical vein endothelial cells. Neutrophils were removed from control subjects and patients in septic shock and were perfused over endothelial cells at rates representing a range of physiologic shear stresses. In an attempt to examine the effects of fibrin deposition on neutrophil-endothelial cell interactions, neutrophils, with and without platelets, were suspended in plasma and serum was removed from patients in septic shock. In addition, blocking monoclonal antibodies against the platelet receptor P-selectin and neutrophil receptor CD11b/CD18, and a platelet glycoprotein IIb/IIIa inhibitor, were incubated with cells suspended in plasma. Phase contrast video microscopy was used to count the number of neutrophils/mm adherent to endothelial cells during cessation of flow. Neutrophil rolling velocity was calculated as the time required for neutrophils to move across a 1-mm field (mm/sec). Leukoaggregation was defined as the number of neutrophils in aggregates (three or more nuclei) across a 1-mm field. MEASUREMENTS AND MAIN RESULTS: Normal neutrophils exposed to plasma from patients with septic shock demonstrated significant increases in aggregation and endothelial cell adherence with associated decreases in neutrophil rolling velocity. These changes were significantly enhanced in the presence of platelets and significantly attenuated in the presence of serum, which is fibrinogen depleted. Preincubation with antibodies to the surface receptors P-selectin, CD11b/CD18, and glycoprotein IIb/IIIa abrogated the changes in neutrophil aggregation, adhesion, and rolling velocity. CONCLUSIONS: These data suggest that platelets and fibrinogen play an important role in mediating neutrophil-endothelial cell adherence in septic shock.

Blood Platelets↗
PubMed1984

[Capillaroscopy and Raynaud's phenomenon].

Nailfold capillary microscopy is a simple and noninvasive screening test for the etiological diagnosis of Raynaud's phenomenon: --dilating capillary dystrophia, with its large capillary loops (megacapillaries), it is a very specific sign of connective tissue disorders, especially systemic sclerosis; --meshed or branched-tree shaped capillaries are neocapillaries, the meaning of which is very often a dermal vasculitis (S. L. E., rheumatoid arthritis, paraneoplastic Raynaud's phenomenon, or even chilblains); --when numerous abnormal capillary loops and hemorrhages are seen without any special pattern, the diagnosis of idiopathic Raynaud's disease is very unlikely and an extensive biological investigation is needed; --nailfold capillaries look like normal in idiopathic Raynaud's disease and, in this case, only a sharp physical examination and a few biological tests are required. Conjunctival angioscopy is a useful complementary test in some cases when nailfold capillaroscopy is unreliable. Capillary video microscopy is an interesting method for further investigations about the physiopathology of Raynaud's phenomenon.

Capillaries↗
PubMed1989

Fluorescence microscopy in three dimensions.

The combination of the specificity provided by fluorescence microscopy and the ability to quantitatively analyze specimens in three dimensions allows the fundamental organization of cells to be probed as never before. Key features in this emergent technology have been the development of a wide variety of fluorescent dyes or fluorescently labeled probes to provide the requisite specificity. High-quality, cooled charge-coupled devices have recently become available. Functioning as nearly ideal imagers or "electronic film," they are more sensitive than photomultipliers and provide extraordinarily accurate direct digital readout from the microscope. Not only is this precision crucial for accurate quantitative imaging such as that required for the ratioing necessary to determine intracellular ion concentrations, but it also opens the way for sophisticated image processing. It is important to realize that image processing isn't simply a means to improve image aesthetics, but can directly provide new, biologically important information. The impact of modern video microscopy techniques (Allen, 1985; Inoué, 1986) attests to the fact that many biologically relevant phenomena take place at the limits of conventional microscopy. Image processing can be used to substantially enhance the resolution and contrast obtainable in two dimensions, enabling the invisible to be seen and quantitated. Cells are intrinsically three-dimensional. This can simply be a nuisance because of limited depth of focus of the microscope or it could be a fundamental aspect of the problem being studied. In either case, image processing techniques can be used to rapidly provide the desired representation of the data. In this chapter we have discussed the nature of image formation in three dimensions and dealt with several means to remove contaminating out-of-focus information. The most straightforward of these methods uses only information from adjacent focal planes to correct the central one. This approach can be readily applied to virtually any problem and with most commonly available image processing hardware to provide a substantially deblurred image in almost real time. In addition to covering more sophisticated algorithms where the utmost in three-dimensional imaging is required, we have developed a method for extremely rapidly and accurately producing an in-focus, high-resolution "synthetic projection" image from a thick specimen. This is equivalent to that produced by a microscope having the impossible combination of a high-NA objective lens and an infinite depth of focus. A variation on this method allows efficient calculation of stereo pairs.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1999

Mathematical model of oxygen transport in the cerebral cortex.

To estimate the magnitude of hyperemia necessary to support oxidative metabolism in the cerebral cortex during functional activation, a mathematical model of O2 transport from capillary to tissue was developed. Radial and axial gradients of O2 pressure in tissue surrounding a single capillary were calculated at normal and increased cerebral metabolic rates for O2. Cone-shaped tissue geometry and nonlinear oxyhemoglobin dissociation were assumed. Local O2 consumption was assumed to be supported with local tissue pO2 greater than 1 mmHg. The distribution of tissue pO2 was also calculated during moderate hypoxemia (paO2=42 mmHg), using experimental values of red blood cell velocity measured in individual capillaries of the rat cerebral cortex using intravital video-microscopy. The model predicted that moderate increases (</=50%) in cerebral O2 consumption were supported by proportional increases in capillary blood flow. Large increases in O2 consumption (50-110%) were supported by disproportional increases in flow. During moderate hypoxemia, average tissue pO2 decreased but oxygen utilization was sustained when capillary flow was increased to a level measured in experiments. The results suggest a proportional relationship between cerebrocortical blood flow and oxygen consumption in the normal physiological range of functional activation.

Animals↗
PubMed1990

Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts.

Using Xenopus egg extracts arrested in interphase or mitosis, we directly observed differences in microtubule dynamics at different stages of the cell cycle. Interphase extracts were prepared from eggs in the first interphase after meiosis. Mitotic extracts were prepared by addition of purified cyclin to interphase extracts. Microtubules were nucleated by the addition of centrosomes and visualized by fluorescence video-microscopy in extracts to which rhodamine-labeled tubulin had been added. We found a striking difference in microtubule dynamics in mitotic versus interphase extracts. Quantitative analysis revealed that the rates of polymerization and depolymerization are similar in interphase and mitosis and that within the spatial and temporal resolution of our experiments the difference in dynamics is due almost entirely to an increase in the frequency of transition from growing to shrinking (catastrophe frequency) in the mitotic extracts.

Animals↗
PubMed1998

Regulation of endothelial cell motility by complexes of tetraspan molecules CD81/TAPA-1 and CD151/PETA-3 with alpha3 beta1 integrin localized at endothelial lateral junctions.

Cell-to-cell junction structures play a key role in cell growth rate control and cell polarization. In endothelial cells (EC), these structures are also involved in regulation of vascular permeability and leukocyte extravasation. To identify novel components in EC intercellular junctions, mAbs against these cells were produced and selected using a morphological screening by immunofluorescence microscopy. Two novel mAbs, LIA1/1 and VJ1/16, specifically recognized a 25-kD protein that was selectively localized at cell-cell junctions of EC, both in the primary formation of cell monolayers and when EC reorganized in the process of wound healing. This antigen corresponded to the recently cloned platelet-endothelial tetraspan antigen CD151/PETA-3 (platelet-endothelial tetraspan antigen-3), and was consistently detected at EC cell-cell contact sites. In addition to CD151/PETA-3, two other members of the tetraspan superfamily, CD9 and CD81/ TAPA-1 (target of antiproliferative antibody-1), localized at endothelial cell-to-cell junctions. Biochemical analysis demonstrated molecular associations among tetraspan molecules themselves and those of CD151/ PETA-3 and CD9 with alpha3 beta1 integrin. Interestingly, mAbs directed to both CD151/PETA-3 and CD81/ TAPA-1 as well as mAb specific for alpha3 integrin, were able to inhibit the migration of ECs in the process of wound healing. The engagement of CD151/PETA-3 and CD81/TAPA-1 inhibited the movement of individual ECs, as determined by quantitative time-lapse video microscopy studies. Furthermore, mAbs against the CD151/PETA-3 molecule diminished the rate of EC invasion into collagen gels. In addition, these mAbs were able to increase the adhesion of EC to extracellular matrix proteins. Together these results indicate that CD81/TAPA-1 and CD151/PETA-3 tetraspan molecules are components of the endothelial lateral junctions implicated in the regulation of cell motility, either directly or by modulation of the function of the associated integrin heterodimers.

Animals↗