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At least 91 records · Page 5Linked to original sources

PubMed1995

Determination of resting free calcium in barnacle muscle using modified aequorins, buffered calcium injections, and simultaneous image-intensified video microscopy.

Knowing the resting free calcium is important in understanding the role of calcium as an intracellular second messenger. We used a bracketing (null) technique with a luminescent calcium indicator, aequorin, microinjection and image-intensification to measure free calcium in single muscle fibres from the barnacle, Balanus nubilus. We injected modified aequorins (recombinant, and hch-) which after a 30 min diffusion gave reasonable resting glows. Subsequent injection of calcium (strongly buffered with either EGTA or BAPTA, 10 mM) increased or decreased the resting glow depending on the free calcium level in the injected buffer solution. This bracketing (null) method is inherently accurate, but mechanical artifacts on calcium injection reduce the accuracy when total light emission is measured. We therefore used image-intensified video-microscopy of the injected region and video processing (Image-1) of artifact-free regions, to greatly improve the consistency. The luminescence in a pre-selected region of the muscle fibre was measured as a function of time during the injection. Solution calciums were chosen so that if the first injection decreased the resting glow, the second increased it, or vice versa, thus bracketing the true resting value. We used two methods to determine the true value bracketed by our injections: (1) a linear interpolation using the fractional changes in luminescence or (2) a power law interpolation assuming a 2.2 or 2.5 power relationship between luminescence and free calcium. Using these methods, we estimated the free calcium level in the lateral depressor fibres of freshly dredged barnacles to be 279 +/- 36 nM (+/- SD), 339 +/- 42 nM, or 352 +/- 45 nM for the linear, 2.2 and 2.5 powers respectively under the conditions of hch-aequorin and BAPTA buffers (using a K'Ca for BAPTA of 3.0 x 10(6) M-1 for our conditions). Recombinant-aequorin gave essentially the same result while EGTA buffers yielded a somewhat higher value but because of influences of pH on the K'Ca for EGTA (taken as 6.7 x 10(6) M-1 for our conditions) was considered less reliable. Minor changes in [Mg2+] upon buffer injection can lead to underestimates of the true resting [Ca2+] by at most 10%. Thus, we estimate the resting free calcium in barnacle muscle fibres to be 300-380 nM.

Aequorin↗

PubMed1988

Detection of surface movements on single smooth muscle cells: digital video microscopy.

The shortening response of isolated single smooth muscle cells from the toad stomach "Bufo marinus", was studied using digital video microscopy. A computer program was developed to rapidly on-line digitize and store successive video images of the cell as it shortened. Single smooth muscle cells were decorated with tiny anionic exchange resin beads which served as markers for surface motion. Through an interactive software routine, the cell's outline and bead images were defined. Given this information, the program determined the beads location along the length of the cell as well as its angular position on the cell surface. The program allowed the investigator to reconstruct three dimensional images of the cell during contraction. Analysis of successive cell images revealed that during cell shortening, beads would rotate on the cell surface. These data were interpreted as evidence for corkscrew-like shortening in single smooth muscle cells.

Animals↗

PubMed1993

Patch-clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy.

A description is given of the implementation of infrared differential interference contrast (IR-DIC) video microscopy to an upright compound microscope. Using the improved resolution offered by IR-DIC a procedure is described for making patch-pipette recordings from visually identified neuronal somata and dendrites in brain slices. As an example of the application of this technique to electrophysiological recordings from small neuronal processes in brain slices we describe whole-cell current-clamp and cell-attached and excised patch-clamp recordings from the apical dendrites of layer V pyramidal neurons in slices of rat neocortex.

Animals↗

PubMed1990

Characterization of low-light-level cameras for digitized video microscopy.

The use of low-light-level video cameras and solid-state image detectors in conjunction with image digitizers for optical microscopy is increasing dramatically as more people learn about such systems, and as they become more powerful, less expensive, and easier to use. However, there is currently little information available allowing comparison between cameras, or for determining camera suitability for a given experiment. In this paper, we describe a series of tests designed to characterize the performance of low-light-level cameras. The results of these tests should assist in the selection of appropriate cameras for given video microscopy applications.

Image Processing, Computer-Assisted↗

PubMed1992

Epifluorescent video microscopy (EVM) for platelet-biomaterial interactions: elimination of photoactivation and dye effects.

The use of two intracellular dyes for epifluorescent video microscopy (EVM) in observations of cell-surface interactions is evaluated and discussed. This methodology permits determinations of cell adhesion, detachment and movement at the surfaces of biomaterials in the presence of flow and physiological haematocrit. Two tests, one which examines for the effect of incident light on platelet adhesion and one which checks for sufficient light for accurate observation of cells, have been designed. Evaluations were made of the adhesion of platelets labelled with the fluorescent dyes mepacrine and acridine orange, used singly and in combination. The use of a number of light level-dye level combinations with glass and several polymers and the addition of a plasma level of fibrinogen did not show any photoactivation effects. This methodology paves the way for longer than previous exposures to light with our system, from 1 min up to 30 min now. Washed platelet suspensions are preferred; these allow for the selective labelling of specific cells and the removal of dye from the surface of the cell.

Acridine Orange↗

PubMed1998

Quantitative video microscopy.

Obviously there are many variations and embellishments on the topic of quantitating your image. I have tried here to offer you a very practical guide which highlights some of the critical issues paying particular attention to problems which can prevent or compromise your success. These include: errors due to aspect ratio, use of automatic camera settings, improper setting of dynamic range, and use of integer arithmetic. Further information can be found in several other chapters in this volume as well as in the references below. Additionally you shouldn't overlook the technical manuals which come with your video systems. The field of video microscopy has evolved as a cooperative effort between academia and industry. As a result you will find that many of the technical support personnel from microscope, video, and image processing companies are well versed on the issue of video imaging in biology and are more than willing to assist you.

Image Enhancement↗

PubMed1987

Analysis of polarization and orientation of human polymorphonuclear leukocytes by computer-interfaced video microscopy.

Chemotactic behavior is a complex cellular response to chemical environmental stimuli. For polymorphonuclear leukocytes (PMNs), such behavior involves net migration as well as changes in cell shape and cell orientation. Accordingly, we have applied computer-interfaced video microscopy to analyze cell shape and orientation in control and patient PMNs migrating under agarose. From a digitized tracing of the PMNs at the leading front of migration, cells were characterized in terms of area, circumference, and longest dimension. A shape factor and angle of orientation were computed. Numerical shape factors discriminated three PMN morphologies: polar, apolar, and hyperpolar. Only polar cells could be oriented. Orientation of polar cells was defined as toward, away, or disoriented with respect to the chemotactic gradient. Apolar cells were considered to be nonoriented. Of PMNs from healthy controls, 30 +/- 5% of the cells were oriented toward and 11 +/- 4% of the cells were oriented away from the gradient. For PMNs from patients with localized juvenile periodontitis, a 40% deficit in net migration was associated with reduced orientation toward (5 +/- 2%) and elevated orientation away from the gradient (33 +/- 9%). PMNs from a panel of patients with thermal injury showed reduced migration and orientation toward the gradient associated with elevated percentages of apolar cells. Such analysis of PMN polarization and orientation of the leading front permitted calculation of a chemotactic behavior index. Application of this multiparameter index to the analysis of the chemotactic response may identify PMNs that are defective, but not by evaluation of any single variable.

Burns↗

PubMed1995

Video microscopy of cerebrocortical capillary flow: response to hypotension and intracranial hypertension.

Although autoregulation of cerebral blood flow is well established, the response of cerebral capillary circulation to reduced cerebral perfusion pressure (CPP) is unclear. The objective of this study was to determine whether red cell flow velocity in individual capillaries of the cerebral cortex is maintained during acute decreases in CPP. Microcirculation of the superficial parietal cerebral cortex of adult barbiturate-anesthetized artificially ventilated rats was visualized using a new design of closed-perfused cranial window and epifluorescent-intensified video microscopy. Fluorescein-isothiocyanate-labeled red blood cells (FRBC) injected intravenously were used as markers of capillary flow. CPP, defined as mean arterial pressure minus intracranial pressure, was reduced by controlled hemorrhage or by stepwise elevation of local intracranial pressure. The movement of FRBC in the parenchymal capillary network was video recorded at each pressure level, and FRBC velocity in each capillary was measured off-line with use of the dual-window digital cross-correlation technique. FRBC flux in the capillaries was measured by automated cell counting. FRBC velocity at normal perfusion pressure was 1.47 +/- 0.58 (SD) mm/s and changed little in the perfusion pressure range of 70-120 mmHg. The autoregulatory index in this pressure range was 0.0049 mm.s-1.mmHg-1. Opening of previously unperfused capillaries was not observed. FRBC flux correlated with FRBC velocity, but the latter was maintained in a narrower range than FRBC flux, suggesting a decrease in capillary diameter or hematocrit with decreasing perfusion pressure. The results suggest that flow autoregulation is associated with the maintenance of capillary flow velocity and that capillary recruitment does not contribute to flow autoregulation in the rat cerebral cortex.

Animals↗

PubMed1994

Semi-automated measurement of true chord length distributions and moments by video microscopy and image analysis.

The distribution of the lengths of airspace chords in pulmonary parenchyma characterizes many architectural features of the alveoli and alveolar ducts. Laborious to obtain manually, the distributions and density functions may be acquired semi-automatically by video microscopy, digitization and image processing. The accuracy of the estimation is influenced by the microscopical methods and also by the techniques used (i) to convert the digitized greyscale picture to a two-valued image, (ii) to collect the chord lengths and (iii) to compensate for finite field widths. The last problem arises because some chords are completely visible within a field while others are only partially seen, since one of the two air-tissue boundaries lies outside the field of view. This error systematically biases the observed distribution. This paper contains solutions to hardware, software and analytic problems encountered while developing the capability to measure airspace chord length density functions semi-automatically. Formulas for estimating the true chord length density function from samples of observed chord lengths are presented. Also given are formulas for the estimation of the first and second moments of the true chord length distribution from the means of observed chord lengths. These techniques of image preparation and analysis should be suitable for characterizing particle, grain or cell size distributions, especially where many profiles fall partially outside the field of view.

Animals↗

PubMed2000

Investigation of the hydrolysis of single DNA molecules using fluorescence video microscopy.

We developed a method of studying the hydrolysis of single DNA molecules by exonuclease (exo) III using fluorescence video microscopy. A single DNA molecule immobilized at one end on a coverslip and labeled with a fluorescent bead at the other end confined the motion of a bead to a two-dimensional projected circular area determined by the contour length of DNA. The radius of this area decreased with time after the addition of exo III, Mg2+, or a single-stranded DNA-binding protein (SSB), which caused the single-stranded (ss) DNA to twist around itself. However, the radius was relatively constant over time in the absence of Mg2+, which is a cofactor in exo III activity, even when exo III and SSB were both present. These observations indicated that the decrease in the radius was due to hydrolysis of DNA by exo III. We then evaluated the rate of exo III hydrolysis of single DNA molecules by monitoring the decrease in the radius.

DNA↗

PubMed1986

Blood platelet surface interactions on fibrinogen under flow as viewed with fluorescent video-microscopy.

The interaction of fluorescently labeled blood platelets with fibrinogen-coated glass was studied in Poiseuille flow at 3 wall shear rates, 40, 80 and 944 s-1. Observations were made via video-microscopy at a distance of 0.5 cm from a tube's entrance over a 1370 microns 2 portion of luminal area. The rates of arrival and detachment, and the net rate of adhesion of cells increased nonlinearly with flow rate. The fraction of arriving cells, first contacts, which adhered without subsequent movement and the fraction of arriving cells which adhered, moved to new positions and then remained adherent, were maximal at 80 s-1. For platelets which adhere and then move to a number of new positions, the likelihood of permanent adhesion is greater than 85 percent. The adhesion process is one in which 40-60 percent of cells permanently adhere on first contact with an additional 30 percent adhering after several moves along the surface. Cells contacting where a platelet was previously adherent had a greater chance of adhering than they would on an unaltered fibrinogen surface. The efficiency of platelet adhesion is greater for second contacts than for first contacts on unaltered fibrinogen coated surface.

Analysis of Variance↗

PubMed1986

Platelet adhesion to fibrinogen-coated glass at an abrupt tubular expansion viewed with fluorescent video-microscopy.

The adhesion and detachment of human washed platelets was studied on the surface of the larger tube of a tubular expansion. Measurements were made within the vortex, at the reattachment point and downstream of the vortex. Fluorescent video-microscopy of mepacrine labelled platelets was used to record data continuously. Flow was from the smaller to the larger tube at Reynolds numbers (based on upstream conditions) of 75.4 and 212.2. Measurements of the adhesion efficiency for initially contacting cells and an overall adhesion efficiency were made. These efficiencies decreased with increasing Reynolds number. There was a pattern of variability for both efficiencies with respect to position and Reynolds number which is consistent with the generation of the unstable flow at the reattachment point.

Analysis of Variance↗

PubMed2006

In vitro continuous amperometry with a diamond microelectrode coupled with video microscopy for simultaneously monitoring endogenous norepinephrine and its effect on the contractile response of a rat mesenteric artery.

Continuous amperometry with a diamond microelectrode and video microscopy were used to record (in vitro) endogenous norepinephrine release simultaneously with the evoked contractile response of a mesenteric artery from a healthy Sprague Dawley rat. Norepinephrine (NE) is a vasoconstricting neurotransmitter released from sympathetic nerves that innervate the smooth muscle cell layers surrounding arteries and veins. Using these two techniques along with several drugs, the NE released at sympathetic neuroeffector junctions nearby the microelectrode was measured as an oxidation current. Key to the amperometric measurement was the use of a diamond microelectrode because of the response sensitivity, reproducibility, and stability it provided. NE release was elicited by electrical stimulation at frequencies between 1 and 60 Hz, with a maximum response seen at 20 Hz. Confirmation that the oxidation current was, in fact, associated with endogenous NE came from the results of several drugs. Tetrodotoxin (TTX, 0.3 microM), a voltage-dependent sodium channel antagonist that blocks nerve conduction, abolished both the oxidation current and the arterial constriction. The alpha(2)-adrenergic autoreceptor antagonist, yohimbine (1.0 microM), caused an increase in the oxidation current and the corresponding constriction. The addition of cocaine (10 microM), an antagonist that inhibits neuronal NE reuptake, caused both the oxidation current and the contractile response to increase. These results, combined with the fact that the hydrodynamic voltammetric E(1/2) for endogenous NE was identical to that for a standard solution, confirmed that the oxidation current was due to NE and that this compound caused, at least in part, the contractile response. The results demonstrate that continuous amperometric monitoring of NE with a diamond microelectrode and video imaging of vascular tone allow real time local measurement of the temporal relationship between nerve-stimulated NE release and arterial constriction.

Animals↗

PubMed1997

Computer-Assisted Laser Scanning and Video Microscopy for Analysis of Cryptosporidium parvum Oocysts in Soil, Sediment, and Feces.

A computer-assisted laser scanning microscope equipped for confocal laser scanning and color video microscopy was used to examine Cryptosporidium parvum oocysts in two agricultural soils, a barnyard sediment, and calf fecal samples. An agar smear technique was developed for enumerating oocysts in soil and barnyard sediment samples. Enhanced counting efficiency and sensitivity (detection limit, 5.2 x 10(sup2) oocysts(middot)g [dry weight](sup-1)) were achieved by using a semiautomatic counting procedure and confocal laser scanning microscopy to enumerate immunostained oocysts and fragments of oocysts in the barnyard sediment. An agarose-acridine orange mounting procedure was developed for high-resolution confocal optical sectioning of oocysts in soil. Stereo images of serial optical sections revealed the three-dimensional spatial relationships between immunostained oocysts and the acridine orange-stained soil matrix material. In these hydrated, pyrophosphate-dispersed soil preparations, oocysts were not found to be attached to soil particles. A fluorogenic dye permeability assay for oocyst viability (A. T. Campbell, L. J. Robertson, and H. V. Smith, Appl. Environ. Microbiol. 58:3488-3493, 1992) was modified by adding an immunostaining step after application of the fluorogenic dyes propidium iodide and 4(prm1),6-diamidino-2-phenylindole. Comparison of conventional color epifluorescence and differential interference contrast images on one video monitor with comparable black-and-white laser-scanned confocal images on a second monitor allowed for efficient location and interpretation of fluorescently stained oocysts in the soil matrix. This multi-imaging procedure facilitated the interpretation of the viability assay results by overcoming the uncertainties caused by matrix interference and background fluorescence.

Journal Article↗

PubMed2000

Dynamics of the mitochondrial reticulum in live cells using Fourier imaging correlation spectroscopy and digital video microscopy.

We report detailed studies of the dynamics of the mitochondrial reticulum in live cells using two independent experimental techniques: Fourier imaging correlation spectroscopy and digital video fluorescence microscopy. When both methods are used to study the same system, it is possible to directly compare measurements of preaveraged statistical dynamical quantities with their microscopic counterparts. This approach allows the underlying mechanism of the observed rates to be determined. Our results indicate that the dynamics of the reticulum structure is composed of two independent contributions, each important on very different time and length scales. During short time intervals (1-15 sec), local regions of the reticulum primarily undergo constrained thermally activated motion. During long time intervals (>15 sec), local regions of the reticulum undergo long-range "jump" motions that are associated with the action of cytoskeletal filaments. Although the frequency of the jumps depend on the physiological state of the cells, the average jump distance ( approximately 0.8 microm) is unaffected by metabolic activity. During short time intervals, the dynamics appear to be spatially heterogeneous, whereas the cumulative effect of the infrequent jumps leads to the appearance of diffusive motion in the limit of long time intervals.

Biophysical Phenomena↗

PubMed1987

Direct measurements of cell number using computer-aided video microscopy.

Quantitative studies in cell culture require accurate measurements of cell density and kinetics. We have developed a direct, rapid, and noninvasive method for measuring cell number in monolayer culture. Using computer-aided video microscopy, cell number was measured without detaching or chemically destroying the cells, thereby allowing sequential measurements in the same cell population. Cell number measured by computer-aided microscopy closely correlated with hemocytometer counts and determinations of total cell protein. For high-density monolayers of mesenchymal cells, however, staining was required for accurate counts. Unlike other techniques for measuring cell density, computer-aided microscopy was especially accurate in medium- to low-density cultures (less than 6000 cells/cm2). In addition, we applied this technique to the construction of separate proliferation curves for glomerular mesangial and vascular endothelial cells in coculture. These measurements by cell type in coculture are impossible using conventional methods for determining cell number.

Animals↗

PubMed2000

High-speed video microscopy and computer enhanced imagery in the pursuit of bubble dynamics.

The equipment and method for studying transient bubble dynamics are described in simple sonochemical reactors and presented using still frames from high-speed video microscopy (500 fps). Effects on aeration bubbles (mean size 1-3 mm diameter) and the cavitation induced species (< 0.5 mm diameter) are studied. The images are computer enhanced to improve interpretation of such features as the maximum ellipsoidal distortion at the nodal sound plane and spherical shape regain with due consideration of energy involved and expansion effects at the nodal sound plane. Also immersion depth/pressure effects, as the bubbles transcend the sound field column, in the cylindrical reactor, are recorded for evaluation of nodal and antinodal sound wave effects. Positions of the nodal and antinodal regions are marked using a novel tungsten halogen bulb technique and verified using the sonoelectroluminescent approach with the classical luminol/hydrogen peroxide chemistry which is enhanced under the sound field conditions.

Journal Article↗

PubMed1993

Cell and sarcomere contractile performance from the same cardiocyte using video microscopy.

The relationship between whole cell and sarcomere contractile performance from within the same myocyte remains unclear. In the present study, the dynamic properties of whole cell and sarcomere contractile performance were examined from the same myocyte by computer-assisted video microscopy. Isolated canine left ventricular myocytes were field stimulated at 1 Hz, and whole cell and sarcomere contractile performance was measured in the unloaded unattached state (n = 16) and after attachment to a basement membrane substrate (n = 18). Whole cell and sarcomere contractile measurements were obtained immediately on initiation of electrical stimulation as well as at steady state, after which measurements were repeated in the presence of 25 nM isoproterenol. Video-microscopic images of whole cell and sarcomere contractions were obtained at final magnifications of x1,100 and x5,500, respectively. By use of a 240-Hz high-scan-rate charge-coupled device camera and a video-based edge-detection system synchronized with the camera video output, the myocyte and sarcomere motion data were digitized. Steady-state percentage and velocity of shortening for whole cells and sarcomeres were 4.75 +/- 0.30% and 56.50 +/- 2.37 microns/s and 8.63 +/- 0.60% and 2.24 +/- 0.46 microns/s, respectively, for the attached myocytes and 8.63 +/- 0.48% and 71.38 +/- 6.14 microns/s and 11.73 +/- 3.22% and 2.72 +/- 0.62 microns/s, respectively, for the unattached myocytes. With the initiation of electrical stimulation, the extent of the shortening-velocity of relengthening relationship increased in a linear fashion for the attached (whole cell, r = 0.87; sarcomere, r = 0.90; both P < 0.001) and unattached myocytes (whole cell, r = 0.83; sarcomere, r = 0.88; both P < 0.001). In all experiments, isoproterenol significantly increased the slope of these linear relationships (P < 0.01). Furthermore, the relationship between whole cell and sarcomere velocity of shortening was highly linear (r > 0.91, P < 0.001). In summary, this study demonstrated that the video-based edge-detection technique could be adapted to measure cell and sarcomere contractile performance from the same myocyte. Furthermore, a significant linear relationship exists between whole cell and sarcomere contractile dynamics with alterations in both load and inotropic state.

Animals↗