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At least 145 records · Page 8Linked to original sources

Time-lapse video microscopy of gliding motility in Toxoplasma gondii reveals a novel, biphasic mechanism of cell locomotion.

Toxoplasma gondii is a member of the phylum Apicomplexa, a diverse group of intracellular parasites that share a unique form of gliding motility. Gliding is substrate dependent and occurs without apparent changes in cell shape and in the absence of traditional locomotory organelles. Here, we demonstrate that gliding is characterized by three distinct forms of motility: circular gliding, upright twirling, and helical rotation. Circular gliding commences while the crescent-shaped parasite lies on its right side, from where it moves in a counterclockwise manner at a rate of approximately 1.5 microm/s. Twirling occurs when the parasite rights itself vertically, remaining attached to the substrate by its posterior end and spinning clockwise. Helical gliding is similar to twirling except that it occurs while the parasite is positioned horizontally, resulting in forward movement that follows the path of a corkscrew. The parasite begins lying on its left side (where the convex side is defined as dorsal) and initiates a clockwise revolution along the long axis of the crescent-shaped body. Time-lapse video analyses indicated that helical gliding is a biphasic process. During the first 180(o) of the turn, the parasite moves forward one body length at a rate of approximately 1-3 microm/s. In the second phase, the parasite flips onto its left side, in the process undergoing little net forward motion. All three forms of motility were disrupted by inhibitors of actin filaments (cytochalasin D) and myosin ATPase (butanedione monoxime), indicating that they rely on an actinomyosin motor in the parasite. Gliding motility likely provides the force for active penetration of the host cell and may participate in dissemination within the host and thus is of both fundamental and practical interest.

Animals↗

Dynamic, real time imaging of ion activities in single living cells using fluorescence video microscopy and image analysis.

Quantitative Fluorescence Microscopy is a powerful analytical tool for the in vivo determination of molecules and ions concentrations in cell populations. Until recently the results obtained represent the average value from a large number of cells under the microscope. The recent development of ratiometric ion-sensitive fluorochromes has provided us with a molecular stopwatch, with which we can monitor microscopic and submicroscopic cellular events in individual cells. These probes can be introduced into the cytoplasm of living cells and as the ionic concentration of, i.e., calcium and hydrogen in the cell interior changes, the dyes undergo changes in fluorescence consisting of wavelength shifts and quantum efficiency. Combining this powerful new technique with ultra-sensitive low-light-level video cameras and digital image processing to the fluorescence microscope permits the study of both spatial and temporal distribution of ions localized on or within intact single cells. Details of the procedures and the equipment required for such a fluorescence ratio imaging system are described. A review of some of the fluorescent probes used for measuring intracellular free Ca2+ and pH will be illustrated with examples of data obtained with a Digital Image Processing System in our laboratory. The use of quantitative video fluorescence microscopy is proposed as a means of studying intracellular chemical pathology both for the dynamic investigations of intracellular processes and for diagnosis of diseases.

3T3 Cells↗

Neurotransmitter-induced exocytosis in goblet and acinar cells of rat nasal mucosa studied by video microscopy.

To investigate the mechanism and neural control of the nasal secretion, we observed the isolated rat nasal mucosa by video-enhanced differential interference contrast microscopy. This technique allowed us to visualize abrupt changes of the individual granules leading to degranulation in the acinar cells and in epithelial goblet cells during secretory stimulation. This image provided evidence that exocytosis is the major mode for regulated secretion in the nasal acinar cells and goblet cells. Acetylcholine (ACh, 0.1-100 microM), substance P (SP, 0.1-10 microM), and vasoactive intestinal peptide (VIP, 0.1-1 microM) induced exocytotic responses and shrinkage of the acinus in a concentration-dependent manner. The effects of ACh (10 microM) on the acinus were clearly inhibited by atropine (5 microM), but the effects of SP (1 microM) and VIP (1 microM) were not. The acinar shrinkage always started before exocytosis, suggesting that the fluid secretion precedes the mucus release. In goblet cells, SP (1 microM) and ACh (10 microM) increased the frequency of exocytotic responses significantly, suggesting that these substances truly play the role of a neurotransmitter for nasal secretion. Histamine (HIST) induced no visible response. The effect of HIST on secretory cells may be neuronally mediated in vivo.

Acetylcholine↗

Computer detection of the rapid diffusion of fluorescent membrane fusion markers in images observed with video microscopy.

We have developed an algorithm for automated detection of the dynamic pattern characterizing flashes of fluorescence in video images of membrane fusion. The algorithm detects the spatially localized, transient increases and decreases in brightness that result from the dequenching of fluorescent dye in phospholipid vesicles or lipid-enveloped virions fusing with a planar membrane. The flash is identified in video images by its nonzero time derivative and the symmetry of its spatial profile. Differentiation is implemented by forward and backward subtractions of video frames. The algorithm groups spatially connected pixels brighter than a user-specified threshold into distinct objects in forward- and backward-differentiated images. Objects are classified as either flashes or noise particles by comparing the symmetries of matched forward and backward difference profiles and then by tracking each profile in successive difference images. The number of flashes identified depends on the brightness threshold, the size of the convolution kernel used to filter the image, and the time difference between the subtracted video frames. When these parameters are changed so that the algorithm identifies an increasing percentage of the flashes recognized by eye, an increasing number of noise objects are mistakenly identified as flashes. These mistaken flashes can be eliminated by a human observer. The algorithm considerably shortens the time needed to analyze video data. Tested extensively with phospholipid vesicle and virion fusion with planar membranes, our implementation of the algorithm accurately determined the rate of fusion of influenza virions labeled with the lipophilic dye octadecylrhodamine (R18).

Algorithms↗

New methods for cytotoxicity testing: quantitative video microscopy of intracellular motion and mitochondria-specific fluorescence.

The aim of this study is to qualify the application the new microscopic methods fluorescence and AVEC-DIC (Allen video-enhanced contrast differential interference contrast) microscopy for toxicity testing. The effects of 2-OH-ethyl methacrylate (HEMA), a toxic acrylic monomer, on human fibroblasts was tested. The HEMA concentrations used were 0.01-1% at incubation times of 1-24 h. The cells were observed with AVEC-DIC microscopy and fluorescent staining to evaluate the velocity of lysosomal movement, the number and morphology of the mitochondria, and the fine structure of the cell. In the samples treated with the toxic compound the lysosomal movement changed, as did the morphology of the mitochondria and of the whole cells. The results are compared and discussed with regard to the results of conventional cytotoxicity tests performed in parallel. The new methods proved to be more sensitive and yielded more specific information on the cellular changes caused by the compound.

Cell Movement↗

Mitochondrial fluorescence patterns in rhodamine 6G-stained myocardial cells in vitro. Analysis by real-time computer video microscopy and laser microspot excitation.

Cellular fluorescence in vitro has been studied employing a low light-level video system interfaced with a real-time image array-processing computer system. Changes in cytoplasmic (mitochondrial) fluorescence in myocytes employing the probe rhodamine 6G have been studied over real time with the aid of several computer-based programs. An oscillating pattern of fluorescence is observed that appears to reflect localized variations in mitochondrial activity. The low light level video computer system used in this study compares favorably with laser-stimulated microspot fluorescence photon-counting techniques for the detection of subcellular fluorescence.

Animals↗

Measurement of ciliary beat frequency using high-speed video microscopy.

Studies on the ciliary beat frequency (CBF) of frog palate epithelium are described. A high-speed video microscope system was used to record ciliary activity at 60 and 180 fields per second (fps). Temperature-controlled experiments were facilitated by a specially designed observation chamber. A total of 1150 measurements were performed on intact tissue (edge and surface observations) and single cells. The overall mean values of CBF at 60 fps (18.8 Hz) and 180 fps (19.1 Hz) were statistically indistinguishable (p less than 0.05). A temperature variation of 0.235 Hz/degree C was obtained for the range 24-27 degrees C. Comparison of results by two observers at both field rates indicated no statistical differences. The variation of CBF with time after collection was also not significant. It was determined that measurements obtained from the edge and single-cell recordings were preferable to those obtained from surface observations.

Animals↗

Acrosomal reaction of thyone sperm. I. Changes in the sperm head visualized by high resolution video microscopy.

Structural changes inside the head of Thyone sperm undergoing the acrosomal reaction were followed with a high-resolution, differential interference contrast (DIC) video microscope. The beating sperm, adhering by their midpiece to the cover slip of a wedge perfusion chamber, were activated by a calcium ionophore (20 microM A23187) suspended in sea water containing 50 mM excess CaCl2. Before activation of the sperm, the acrosomal region appears as a 1.1-microM diameter sphere, slightly less dense than the rest of the sperm head. Upon activation, the acrosome pops; the acrosomal region suddenly swells and its refractive index drops. After approximately 1 s, a crescent-shaped periacrosomal cup appears behind the acrosomal vacuole. In the next several seconds, the cup loses more refractive index and expands forward as the acrosomal process extends. The acrosomal vacuole becomes smaller, but without appreciable drop in refractive index. These observations, coupled with the behavior of the extending acrosomal process reported in the companion paper, and in electron microscopy (EM) and early physiological studies, suggest that the acrosomal process is extended by a combination of the explosive polymerization of actin and the osmotic swelling of the periacrosomal cup material. In this paper, we also consider the meaning of the enhanced DIC image seen in the high-resolution video microscope, and discuss the reliability of measurements on small linear dimensions made with the DIC microscope.

Acrosome↗

Measurement of material extravasation in microvascular networks using fluorescence video-microscopy.

We have developed a new method using fluorescence videomicroscopy to quantitate the extravasation of intravenously injected materials. This method can measure the relative plasma concentration of, and the vascular permeability to, these materials in microcirculatory preparations which contain multiple blood vessels in a field of view. The image of a tissue area containing multiple blood vessels is recorded via a SIT camera immediately before, and for an extended period after, the intravenous injection of a bolus of fluorescent test tracers. The videotape is analyzed off-line. At various time points, the light intensities of the entire tissue area and of several spots over selected vessels are measured. These measurements are then used to calculate the fluorescent light intensities arising from the tracers inside vessels (Iv) and in the interstitial region (Ii). Iv represents the relative amount of the tracers in the plasma, and Ii represents that in the interstitium. Iv and Ii are used to calculate an average permeability (P) for the vessels in the observed region. The benefit of this method is that it can be used to compare permeability of various tissues of interest or to serially evaluate changes in P in the same tissue over time. In this study, it was applied to measuring P to albumin as well as to liposomes in granulating and implanted tumor tissues in a rat skin flap window chamber. Changes in permeability to a small molecule (sulforhodamine B) before and during bradykinin application were also measured. The results of these experiments indicate that the relative plasma concentrations predicted by this method conformed well to those measured directly from blood samples, and the measured permeability values were consistent with previously published data. Therefore, this method provides a valid approach for quantitatively measuring the extravasation of intravenously injected molecular and colloidal materials in microcirculatory preparations. The method has a set of defined experimental conditions and assumptions that cannot be violated, however, or erroneous results can be obtained.

Animals↗

Simultaneous measurements of cytosolic pH and calcium interactions in bovine lactotrophs using optical probes and four-wavelength quantitative video microscopy.

The properties of pH and calcium homeostasis have been investigated in single bovine lactotrophs by the use of the fluorescent indicators 2',7'-bis(carboxyethyl)-carboxyfluorescein (BCECF) and fura-2 respectively. A method of simultaneous recording from both dyes loaded in the same cell was used. Despite slight crosstalk between the two dyes, physiologically relevant information about the interrelationship between pH and calcium homeostasis was obtained. Three types of interactions were recorded. First, an increase in calcium due to the discharge of intracellular stores by thyrotropin-releasing hormone resulted in no change in cytosolic pH. Secondly, alkalinization by the addition of a weak base, NH4Cl, induced a large transient (around 1000 nM) and a small (a few tens of nanomoles per liter) sustained increase in cytosolic calcium. The former is partly due to release from agonist-sensitive stores. Thirdly, upon the removal of NH4Cl the cytoplasm became acidic, which induced a release of calcium from intracellular stores in some cells. In addition we demonstrate that the recovery from acid load is sensitive to extracellular Na+, suggesting the presence of Na+/H+ exchange mechanisms in bovine lactotrophs. Interestingly we have also found that, at rest, removal of Na+ from the bathing medium results in a decrease in resting [Ca2+]i, paralleled by a reduction in pHi. This suggests a role for Na+/H+ exchange in determining resting [Ca2+]i.

Ammonium Chloride↗

Acrylamide and glycidamide impair neurite outgrowth in differentiating N1E.115 neuroblastoma without disturbing rapid bidirectional transport of organelles observed by video microscopy.

The nature of the pathogenic insult in acrylamide neuropathy is unknown, but axonal transport disturbances are suspected. Using N1E.115 neuroblastoma in vitro, we examined acrylamide and related compounds in terms of general cytotoxicity, ability to block neurite outgrowth, and effects on neurite integrity and fast axonal transport. Acrylamide, glycidamide, and methylene-bis-acrylamide were weakly cytotoxic in a 51Cr-release assay, but only at > or = 10 mM (order of efficacy: methylene-bis-acrylamide > glycidamide > acrylamide). Neurite outgrowth by differentiating cells was inhibited at 100-fold lower concentrations, with similar EC50 values for all three toxicants, i.e., acrylamide, 70 +/- 15 microM; methylene-bis-acrylamide, 92 +/- 31 microM; glycidamide, 120 +/- 30 microM. Only glycidamide (1 mM) caused degeneration of established neurites within a period of 48 h. Video-enhanced contrast differential interference contrast microscopy was used to test the effect of acrylamide and glycidamide on organelle transport in the neurites. In exposures of < or = 48 h at 1 mM, neither toxicant altered bidirectional organelle flux, measured as organelles transported per minute per micrometer of neurite diameter. Anterograde and retrograde organelle speeds were also undisturbed. These results suggest that mechanisms other than direct inhibition of organellar motility are responsible for acrylamide's neurotoxicity in vivo.

Acrylamide↗

[Examination of the cornea of intact donor eyes with confocal slit-scanning video microscopy].

The specular microscopic examination of the endothelium of intact donor eyes can be rendered difficult or even impossible due to light scattering of stromal edema. We have been able to visualize large areas of the corneal endothelium of a donor eye with advanced stromal edema--stored for over 32 h. in a moist chamber--with a confocal slit-scanning video microscope. Furthermore, this optical arrangement enables the examination of very thin tissue sections with high resolution and a remarkably enhanced contrast of the corneal micromorphology (epithelial cells, Bowman's membrane, nerve fibers, keratocytes, endothelial cells).

Alcoholism↗