[Dynamic analysis of neuronal responses by video microscopy: direct visualization of release and acceptance of neuronal signals].
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Animals↗
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[Observation of brain slice using infra-red differential interference contrast (IR-DIC) video microscopy].
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Animals↗
[Analysis of axonal transport using video microscopy].
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Animals↗
Time lapse video microscopy and ultrastructure of penetrating sporozoites, types 1 and 2 parasitophorous vacuoles, and the transformation of sporozoites to tachyzoites of the VEG strain of Toxoplasma gondii.
Videomicroscopy and transmission electron microscopy were used to study the interaction of Toxoplasma gondii sporozoites with cultured cardiopulmonary artery endothelial, embryonic bovine tracheal and Madin-Darby bovine kidney cells. No moving junction or exocytosis of rhoptries, micronemes, and dense granules was detected during the initial penetration of sporozoites into cultured cells, whereas constriction of the sporozoite and partial exocytosis of rhoptries occurred during movement of the sporozoite from the first parasitophorous vacuole (PV1) into the second vacuole (PV2). The PV1 was unusually large, lacked a tubulovesicular membrane network (TMN), and had an indistinct parasitophorous vacuolar membrane (PVM). Comparatively, the PV2 was small, had a distinct PVM, contained a well-developed TMN, and was surrounded by numerous host cell mitochondria. Sporozoites that passed completely through cells carried with them an envelope of host cell membranes and cytoplasm. Cultured cells occasionally endocytosed sporozoites that were enveloped by host cell material. After formation of the PV2, sporozoites replicated by endodyogeny to form tachyzoites.
Animals↗
Maximization of skin capillaries during intravital video-microscopy in essential hypertension: comparison between venous congestion, reactive hyperaemia and core heat load tests.
Intravital capillary video-microscopy is a dynamic method for studying skin capillaries. The technique of direct intravital microscopy (without dyes) depends on the presence of red blood cells inside capillaries for their identification. The aim of the present study was to compare different techniques to try to establish the best method for maximizing the number of visible perfused capillaries during intravital capillary microscopy. We compared the effects of venous congestion with those of post-occlusive reactive hyperaemia (Study 1). We also investigated venous congestion followed first by post-occlusive reactive hyperaemia and then by a core heat load test (Study 2). Finally we investigated venous congestion followed by post-occlusive reactive hyperaemia combined with venous congestion (Study 3). In Study 1, capillary density increased with venous congestion from a baseline value of 74+/-2 (mean+/-S.E.M.) per field to 82+/-3 per field (P<0.0001; analysis of variance). With reactive hyperaemia, there was an apparent decrease in visible capillary density to 69+/-2 per field. In Study 2, baseline capillary density was 69+/-4 per field, and this increased significantly with venous congestion to 74+/-4 per field (P=0.01). With both reactive hyperaemia and core heat load, the apparent density was 62+/-4 per field. In Study 3 the baseline density was 70+/-2 per field, and this increased significantly with venous congestion to 80+/-3 per field (P<0.0001). With reactive hyperaemia combined with venous congestion, the density was 81+/-3 per field (P=0.328 compared with venous congestion alone). The results show that venous congestion at 60 mmHg for 2 min is the most effective method for visualization of the maximal number of perfused skin capillaries during intravital video-microscopy.
Adult↗
Movement of fine particles on an air bubble surface studied using high-speed video microscopy.
A CCD high-speed video microscopy system operating at 1000 frames per second was used to obtain direct quantitative measurements of the trajectories of fine glass spheres on the surface of air bubbles. The glass spheres were rendered hydrophobic by a methylation process. Rupture of the intervening water film between a hydrophobic particle and an air bubble with the consequent formation of a three-phase contact was observed. The bubble-particle sliding attachment interaction is not satisfactorily described by the available theories. Surface forces had little effect on the particle sliding with a water film, which ruptured probably due to the submicrometer-sized gas bubbles existing at the hydrophobic particle-water interface.
Journal Article↗
Simultaneous assessment of red cell perfusion in skeletal muscle by laser Doppler flowmetry and video microscopy.
The objective of this study was to compare temporal and spatial variations of the laser Doppler flowmeter output (V) with the corresponding variations of perfusion (cells/mm3 X mm/s) evaluated by video microscopy. The flowmetry and video microscopy sampled 2 mm3 (approx.) and 0.84 mm3 surface volumes of the sartorius muscle in anesthetized frogs, respectively. The overall ranges of the output and perfusion measurements were from 0.01 to 0.72 V and from 45 to 1404 cells/mm3 X mm/s. Within these ranges, temporal variations induced by muscle contraction correlated well (overall r = 0.91), but the spatial variations associated with the resting state correlated poorly (overall r = 0.45). When the penetration of the laser light was limited to 0.3-0.4 mm (to make the volumes sampled by both techniques more comparable) the overall r of the spatial comparison increased to 0.86. It is concluded that the flowmeter (1) is affected by red cell perfusion below the tissue depth of 0.3-0.4 mm, and (2) can follow both the temporal and spatial variations of red cell perfusion in the tissue examined.
Capillaries↗
Imaging of electrically induced fast motion by video microscopy and triggered flash illumination.
A simple and inexpensive method is described for imaging with video microscopy fast transient events that can be triggered electrically. An electronic system was developed that triggered stroboscopic illumination and generated an electrical step function. The essential feature was precise control of a sub-millisecond time delay between electrical stimulus and the following short pulse of light. With this technique (15 microseconds time resolution, 50 frames/s) different phases of the exocytosis and discharge of nematocysts from Hydra vulgaris can be visualized. It was shown that not only stenoteles, but also desmonemes, can discharge in less than 0.5 ms.
Animals↗
Measuring microlymphatic flow using fast video microscopy.
Despite advances in the measurement of lymphatic function, little is known about the actual velocities of flow in microlymphatic ( approximately 100 mum diam) vessels. In this work, video microscopy and particle tracking methods are adapted and integrated with an ultra-high-speed imaging camera to obtain measurements of lymph velocities throughout the entire lymphatic contraction cycle in the ratmesentery, something that previous systems were incapable of measuring. To determine the system's accuracy, calibration experiments are conducted across the hypothesized physiologically significant range of velocities for microlymphatic flow (up to 15 mmsec). The system shows high accuracy, less than 2% error, when comparing actual with measured velocities. Microspheres flowing through 140-mum-diam tubing are imaged to demonstrate the system's ability to determine flow rates in these small vessels by measuring particle velocities. To demonstrate biological applicability, mesenteric microlymphatics in loops of the small intestine of three male Sprague-Dawley rats are exteriorized and imaged with the high-speed system at a rate of 500 framessec for several contraction sequences. Lymph velocity fluctuates cyclically with the vessel wall contractions, ranging from -1 to 7 mmsec. These rates are higher than would be possible with standard video microscopy (3.75 mmsec maximum).
Animals↗
Direct observation of epicardial coronary capillary hemodynamics during reactive hyperemia and during adenosine administration by intravital video microscopy.
Using high-resolution intravital charge-coupled device video microscopy, we visualized the epicardial capillary network of the beating canine heart in vivo to elucidate its functional role under control conditions, during reactive hyperemia (RH), and during intracoronary adenosine administration. The pencil-lens video-microscope probe was placed over capillaries fed by the left anterior descending artery in atrioventricular-blocked hearts of open-chest, anesthetized dogs paced at 60-90 beats/min (n = 17). In individual capillaries under control conditions, red blood cell flow was predominant during systole or diastole, indicating that the watershed between diastolic arterial and systolic venous flows is located within the capillaries. Capillary flow increased during RH and reached a peak flow velocity (2.1 +/- 0.6 mm/s), twice as high as control (1.2 +/- 0.5 mm/s), with enhancement of intercapillary cross-connection flow and enlargement of diameter (by 17%). With adenosine, capillary flow velocity significantly increased (1.8 +/- 0.7 mm/s). However, the increase in volumetric capillary flow with adenosine estimated from red blood cell velocity and diameter was less than the increase in arterial flow, whereas that during RH was nearly equivalent to the increase in arterial flow. There was a time lag of approximately 1.5 s for refilling of capillaries during RH, indicating their function as capacitance vessels. In conclusion, the coronary capillary network functions as 1) the major watershed between diastolic-dominant arterial and systolic-dominant venous flows, 2) a capacitor, and 3) a significant local flow amplifier and homogenizer of blood supply during RH, but with adenosine the increase in capillary flow velocity was less than the increase in arterial flow.
Adenosine↗
Digital video microscopy for the undergraduate histology laboratory.
An undergraduate histology course was profoundly changed through the introduction of digital video microscopy. Students have access to a networked, Macintosh-based imaging laboratory where they can digitally capture, enhance, analyse, and media output microscope originated images. By increasing the access to image information, students are able to assume a more active inquiry mode in a microscope-based course. The technology allowed students to increase intellectual sharing among themselves and to leave 'digital legacies' for future classes.
Computer Communication Networks↗
Observation of single influenza virus-cell fusion and measurement by fluorescence video microscopy.
We have used intensified video fluorescence microscopy and digital image processing to observe and quantitate influenza virus (A/PR8/34/H1N1) fusion to human erythrocyte membranes. Viruses labeled with the lipid probe octadecylrhodamine B (R18) were seen to undergo fluorescence dequenching and eventual disappearance after exposure to pH levels known to induce virus-cell membrane fusion. Quantitative intensity measurements of single individual particles were possible. From these fluorescence data it has been possible to calculate the fraction of R18 dye molecules transferred from the virus to the cell. The redistribution of the lipid probe upon fusion at pH 5.0 had a t1/2 of 46 s, longer than expected for a free-diffusion model. The R18 loss was approximately twice as fast at pH 5.0 as at pH 5.1. No obvious delay until the start of fluorescence dequenching was observed after the pH changes, suggesting that activation processes are faster than the time resolution, 1-5 s, of the current method.
Erythrocyte Membrane↗
Axonal membrane proteins are transported in distinct carriers: a two-color video microscopy study in cultured hippocampal neurons.
Neurons transport newly synthesized membrane proteins along axons by microtubule-mediated fast axonal transport. Membrane proteins destined for different axonal subdomains are thought to be transported in different transport carriers. To analyze this differential transport in living neurons, we tagged the amyloid precursor protein (APP) and synaptophysin (p38) with green fluorescent protein (GFP) variants. The resulting fusion proteins, APP-yellow fluorescent protein (YFP), p38-enhanced GFP, and p38-enhanced cyan fluorescent protein, were expressed in hippocampal neurons, and the cells were imaged by video microscopy. APP-YFP was transported in elongated tubules that moved extremely fast (on average 4.5 micrometer/s) and over long distances. In contrast, p38-enhanced GFP-transporting structures were more vesicular and moved four times slower (0.9 micrometer/s) and over shorter distances only. Two-color video microscopy showed that the two proteins were sorted to different carriers that moved with different characteristics along axons of doubly transfected neurons. Antisense treatment using oligonucleotides against the kinesin heavy chain slowed down the long, continuous movement of APP-YFP tubules and increased frequency of directional changes. These results demonstrate for the first time directly the sorting and transport of two axonal membrane proteins into different carriers. Moreover, the extremely fast-moving tubules represent a previously unidentified type of axonal carrier.
Amyloid beta-Protein Precursor↗
Time lapse phase contrast video microscopy of directed migration of human microvascular endothelial cells on matrigel.
Migration of microvascular endothelial cells is an early and critical step in angiogenesis. Formation of branching and polygonal cellular aggregates by endothelial cells on matrigel has often been considered to be an in vitro model for angiogenesis, although formation of lumens has not always been confirmed. The dynamics of migration of living cells of a human dermal microvascular endothelial cell line (HMEC-1) on a reconstituted basement membrane matrix have been captured in real time using time lapse video microscopy. The cells exhibit periods of quiescence and directed rapid migration by formation of extensions towards a specific target cell. Cells repeatedly extend flexible protrusions from the cell body both within the plane of the matrix and out of the plane of the matrix into the incubation medium. Connections between protrusions and target cells are made frequently, but not all cells which start to form protrusions achieve connections with other cells. Some of these migrating cells which do not connect arrest before reaching the target, or arrest and retract to their origin. After formation of multicellular polygonal structures, the structures contract to form amorphous clusters of fused cells without visible effects on the underlying matrix. The study demonstrates that time lapse video microscopy is a simple but very useful approach to monitor the dynamics of movements which vary in speed and frequency during migration of living cells.
Basement Membrane↗
A triple electrode for simultaneous investigations of transcutaneous oxygen tension, laser-Doppler flowmetry and dynamic fluorescence video microscopy.
A newly designed triple probe is introduced for measurements of transcutaneous oxygen tension, laser Doppler flowmetry (LDF) and microangiodynamics of skin capillaries by dynamic video microscopy with and without fluorochromes. The performance of the triple probe was checked in 9 healthy volunteers (6 women, 3 men; mean age: 34 years) and 9 patients (5 women, 4 men; mean age: 67 years) with peripheral arterial occlusive disease (PAOD). The mean Doppler ankle/arm pressure ratio was 0.54 +/- 0.30. Six patients suffered from severe claudication, 2 from rest pain and 1 patient had toe and forefoot necrosis. The foot dorsum was selected as measuring site. After recording baseline values of skin surface PO2 (ssPO2) at 37 degrees C, LDF and capillary images, a suprasystolic compression at the ankle level was performed for 4 min. Thirty seconds before cuff opening 0.2 ml/1 l blood volume of 20% sodium fluorescein was injected in an antecubital vein. Sodium fluorescein arrival times, filling times and maximum fluorescent light intensity times were measured, and ssPO2 and LDF were recorded continuously during postocclusive reactive hyperemia (PORH). The results indicate an adequate function of the triple probe. The mean resting ssPO2 was 2.0 +/- 1.9 mm Hg in PAOD patients and 4.0 +/- 3.9 mm Hg in controls (p = 0.185). Maximum ssPO2 during PORH was significantly reduced (p = 0.008) in patients (3.1 +/- 2.1 mm Hg) compared to healthy subjects (11.8 +/- 7.7 mm Hg). Resting LDF values were 6.5 +/- 6.4 perfusion units (PU) in PAOD patients versus 10.3 +/- 8.2 AU in controls (p = 0.295). Peak LDF during PORH was significantly reduced (p = 0.005) in patients (19.5 +/- 6.4 PU) versus healthy subjects (33.8 +/- 11.5 PU.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Analysing functional connectivity in brain slices by a combination of infrared video microscopy, flash photolysis of caged compounds and scanning methods.
We evaluate a novel set-up for scanning functional connectivity in brain slices from the somatosensory cortex of the rat. Upright infrared video microscopy for targeted placement of electrodes is combined with rapid photolysis of bath-applied caged neurotransmitter induced by a xenon flash lamp. Flash photolysis of caged glutamate and electrical stimulation produce comparable field potential responses and demonstrate that the viability of the submerged slices exceeds several hours. Glutamate release leads to field potential responses whose two phases are differentially affected by selective blockade of N-methyl-D-aspartate- and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-type glutamate receptors with DL-2-amino-5-phosphonovaleric acid and 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulphonamide, respectively. Rapid computer-controlled scanning of hundreds of distinct stimulation sites with simultaneous recordings at a fixed reference site allows construction of functional input maps from peak amplitudes and delays to peak of field potential responses. Selective laminar expansion of the functional input maps after bicuculline application demonstrates that the combination of this conveniently assembled set-up with pharmacological and physical manipulations can provide insights into the determinants of functional connectivity in brain slices.
2-Amino-5-phosphonovalerate↗
Tracking of migrating cells under phase-contrast video microscopy with combined mean-shift processes.
In this paper, we propose a combination of mean-shift-based tracking processes to establish migrating cell trajectories through in vitro phase-contrast video microscopy. After a recapitulation on how the mean-shift algorithm permits efficient object tracking we describe the proposed extension and apply it to the in vitro cell tracking problem. In this application, the cells are unmarked (i.e., no fluorescent probe is used) and are observed under classical phase-contrast microscopy. By introducing an adaptive combination of several kernels, we address several problems such as variations in size and shape of the tracked objects (e.g., those occurring in the case of cell membrane extensions), the presence of incomplete (or noncontrasted) object boundaries, partially overlapping objects and object splitting (in the case of cell divisions or mitoses). Comparing the tracking results automatically obtained to those generated manually by a human expert, we tested the stability of the different algorithm parameters and their effects on the tracking results. We also show how the method is resistant to a decrease in image resolution and accidental defocusing (which may occur during long experiments, e.g., dozens of hours). Finally, we applied our methodology on cancer cell tracking and showed that cytochalasin-D significantly inhibits cell motility.
Adenocarcinoma↗