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PubMed1986

The study of fluorescent probes by quantitative video intensification microscopy (QVIM).

In this paper we describe a system for the quantitation and display of fluorescence at the cellular level. It uses a low light level video camera which is interfaced to a fluorescence microscope and to a microprocessor-controlled video digitizing system. With the use of a light pen entry system one can specify areas of the field for measurement. The data obtainable are the area and perimeter of the delimited zone, the distribution of pixel intensities within this zone over a 16-level gray scale, and a value for total fluorescence intensity. Statistical outputs for repeated measurements are also obtained. The system responds linearly to light input, has a high degree of reproducibility, and provides good spatial resolution. Using the DNA-specific dye, Hoechst 33248, in diploid fibroblasts as test material, the system is shown to be able to reproduce expected distributions for amounts of DNA per cell. The capabilities and advantages of pseudocolor display are also demonstrated. We conclude that, in conjunction with appropriate fluorescent probes, systems such as the one described make it possible to do quantitative histochemistry of living cells and to measure substances not previously amenable to study.

DNA↗
PubMed2004

Vessel boundary tracking for intravital microscopy via multiscale gradient vector flow snakes.

Due to movement of the specimen, vasodilation, and intense clutter, the intravital location of a vessel boundary from video microscopy is a difficult but necessary task in analyzing the mechanics of inflammation and the structure of the microvasculature. This paper details an active contour model for vessel boundary detection and tracking. In developing the method, two innovations are introduced. First, the B-spline model is combined with the gradient vector flow (GVF) external force. Second, a multiscale gradient vector flow (MSGVF) is employed to elude clutter and to reliably localize the vessel boundaries. Using synthetic experiments and video microscopy obtained via transillumination of the mouse cremaster muscle, we demonstrate that the MSGVF approach is superior to the fixed-scale GVF approach in terms of boundary localization. In each experiment, the fixed scale approach yielded at least a 50% increase in root mean squared error over the multiscale approach. In addition to delineating the vessel boundary so that cells can be detected and tracked, we demonstrate the boundary location technique enables automatic blood flow velocity computation in vivo.

Algorithms↗
PubMed1995

Activity-related calcium dynamics in lamprey motoneurons as revealed by video-rate confocal microscopy.

In lamprey spinal cord, intracellular calcium ([Ca2+]i) plays a key role in mechanisms regulating neuronal activity in the segmental network for locomotion. In this report, measurements of [Ca2+]i with fluo-3 in various regions of motoneurons in the intact spinal cord were obtained on a high speed confocal microscope following electrical stimulation. Likewise, rhythmic calcium fluctuations within dendrites and axons were seen during "fictive swimming" and were directly correlated with electrical activity. Antidromic stimulation of motoneuron axons induced large calcium transients and revealed spatially restricted "hot spots," both of which required external calcium and were blocked by nickel, but not by known calcium channel antagonists. These results suggest that lamprey spinal cord axons may possess a pharmacologically novel class of calcium channel.

Aniline Compounds↗
PubMed2004

Molecular expressions: exploring the world of optics and microscopy. http://microscopy.fsu.edu.

Our knowledge of the structure, dynamics and physiology of a cell has increased significantly in the last ten years through the emergence of new optical imaging modalities such as optical sectioning microscopy, computer- enhanced video microscopy and laser-scanning microscopy. These techniques together with the use of genetically engineered fluorophores have helped scientists visualize the 3-dimensional dynamic processes of living cells. However as powerful as these imaging tools are, they can often be difficult to understand and fully utilize. Below I will discuss my favorite website: The Molecular Expressions Web Site that endeavors to present the power of microscopy to its visitors. The Molecular Expressions group does a remarkable job of not only clearly presenting the principles behind these techniques in a manner approachable by lay and scientific audiences alike but also provides representative data from each as well.

Cell Physiological Phenomena↗
PubMed2005

Measurement and interpretation of particle-particle and particle-wall interactions in levitated colloidal ensembles.

This paper reports measurements of particle-wall and particle-particle interactions in levitated colloidal ensembles using integrated total internal reflection microscopy (TIRM) and video microscopy (VM) techniques. In levitated colloidal ensembles with area fractions of phiA = 0.03-0.25, ensemble TIRM measured height distribution functions are used to interpret particle-wall interactions, and VM measured pair distribution functions are used to interpret particle-particle interactions using inverse Ornstein-Zernike (OZ) and three-dimensional inverse Monte Carlo (MC) analyses. An inconsistent finding is the observation of an anomalous long-range particle-particle attraction and recovery of the expected Derjaguin-Landau-Verwey-Overbeek (DLVO) particle-wall interactions for all concentrations examined. Because particle-wall and particle-particle potentials are expected to be consistent in several respects, the analytical and experimental methods employed in this investigation are examined for possible sources of error. Comparison of inverse OZ and three-dimensional inverse MC analyses are used to address uncertainties related to dimensionality, effects of particle concentration, and assumptions of the OZ theory and closure relations. The possible influence of charge heterogeneity and particle size polydispersity on measured distribution functions is discussed with regard to inconsistent particle-wall and particle-particle potentials. Ultimately, achieving a consistent understanding of particle-wall and particle-particle interactions in interfacial and confined colloidal systems is essential to numerous complex fluid and advanced material technologies.

Journal Article↗
PubMed2004

Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p.

Exocytosis in the budding yeast Saccharomyces cerevisiae occurs at discrete domains of the plasma membrane. The protein complex that tethers incoming vesicles to sites of secretion is known as the exocyst. We have used photobleaching recovery experiments to characterize the dynamic behavior of the eight subunits that make up the exocyst. One subset (Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo84p) exhibits mobility similar to that of the vesicle-bound Rab family protein Sec4p, whereas Sec3p and Exo70p exhibit substantially more stability. Disruption of actin assembly abolishes the ability of the first subset of subunits to recover after photobleaching, whereas Sec3p and Exo70p are resistant. Immunogold electron microscopy and epifluorescence video microscopy indicate that all exocyst subunits, except for Sec3p, are associated with secretory vesicles as they arrive at exocytic sites. Assembly of the exocyst occurs when the first subset of subunits, delivered on vesicles, joins Sec3p and Exo70p on the plasma membrane. Exocyst assembly serves to both target and tether vesicles to sites of exocytosis.

Actin Cytoskeleton↗
PubMed1995

Delivery to lysosomes in the human carcinoma cell line HEp-2 involves an actin filament-facilitated fusion between mature endosomes and preexisting lysosomes.

We have addressed the following question: what is the mechanism behind the delivery of internalized molecules from mature endosomes to lysosomes in HEp-2 cells, and which role does the cytoskeleton play in this process? Quantitative electron microscopy and immunogold labeling revealed that whereas the cytoskeleton was not of importance for endosome maturation, actin filaments facilitated fusion of mature endosomes with preexisting lysosomes. Delivery to lysosomal degradation was not dependent on protein synthesis as determined biochemically, but was reduced by cytochalasin D. Observations made by electron microscopy as well as by video microscopy of living cells showed that the concerted action of actin filaments and microtubules was responsible for the random distribution and movement of endocytic organelles throughout the cell. Actin microfilaments, however, seem to facilitate perinuclear clustering and frequent fusion of mature endosomes and lysosomes, while microtubules play a role in preventing formation of large lysosome aggregates by separating endosomes and lysosomes and moving them toward the cell periphery. Taken together, our data suggest that delivery of internalized molecules to lysosomal proteolysis takes place by fusion of mature endosomes with preexisting lysosomes and that actin microfilaments somehow facilitate this step.

Actins↗
PubMed2002

Mitochondrial cytochrome c release in radiation-induced apoptosis of human peripheral T cells.

We examined sequential changes in post-irradiated peripheral blood T cells taken from normal volunteers, using a microscopy-video system, mitochondrial membrane potential assay, annexin V, propidium iodide, and cytochrome c ELISA kit. After 5 Gy irradiation with 10 MV X-ray from a linear accelerator, the percentages of apoptotic T cells were estimated as approximately 5, 10, 20, 35, and 70%, at 0, 3, 6, 10, and 20 h after irradiation, respectively, as observed with the microscopy-video system. Using a CCD camera-equipped fluorescence microscope and MitoCapture, a mitochondrial membrane potential indicator, approximately half of the T cells showed dysfunction of mitochondrial membrane potential at 10 h after 5 Gy irradiation. With regard to annexin V and propidium iodide, approximately 40 and 5% of the human peripheral T cells showed positivity against annexin V and propidium iodide at that time, respectively. Mitochondrial cytochrome c release from the mitochondria to the cytosol was confirmed to start at 10 h and to reach a maximum at 20 h after 5 Gy of irradiation. These results demonstrated that mitochondrial cytochrome c release occurred following dysfunction of mitochondrial membrane potential in radiation-induced T cell apoptosis.

Annexin A5↗
PubMed1989

Investigation of the source of the blue field entoptic phenomenon.

The cellular source of the blue field entoptic phenomenon was investigated in two microvascular preparations using video-microscopy with lighting conditions similar to those under which the entoptic phenomenon is visualized within the human eye. In the wing of the hibernating bat, microvascular flow was simultaneously videotaped under transmission illumination at 430 nm and under unfiltered illumination. In the rat cremaster alternating observations were made using transmission illumination at 430 nm and epi-illumination fluorescence microscopy with leukocytes rendered fluorescent by intravenous Quinacrine. In both preparations, low magnification video-microscopy using 430 nm illumination produced a field of particles, which were brighter than the background, flowing within a network of dark vessels. The appearance of the particles and their movement simulated the blue field entoptic particle motion. Under higher magnification, the particles appeared brighter than the plasma gaps between red blood cells and were demonstrated to be leukocytes by morphology, by specific staining and by typical behavioral movement. The particles were observed in terminal arteriols capillaries, and post-capillary venules where they were not obscured by red blood cells. The results of this study of two microvascular preparations strongly suggest that in the human eye the blue field entoptic phenomenon is produced by leukocytes flowing within the macular retinal microvasculature.

Animals↗
PubMed1988

Direct observation of mitotic spindle elongation in vitro.

Successful reactivation in vitro of anaphase B has recently been achieved with mitotic spindles isolated from the diatom Stephanopyxis turris. When a population of isolated spindles was studied indirectly by using immunofluorescence, nearly all of them were found to have elongated; however, when studied directly by using video microscopy, only a small proportion of spindles elongated. We report here conditions that allow nearly all of the spindles to elongate when observed directly with video microscopy. These direct observations validate previous ones made using indirect immunofluorescence. In addition, we find that the isolated spindles elongate with a linear rate, that the elongation is unchanged after the chromatin surrounding the spindles is digested with DNase I, and that during elongation a phase-dense matrix may accumulate in the spindle midzone.

Anaphase↗
PubMed1997

Static image analysis of skin specimens: the application of telepathology to frozen section evaluation.

Although the ability to transmit high-resolution images of histopathological sections could have a profound impact on the practice of pathology, the application of video microscopy to the daily activities of surgical pathology has not been rigorously evaluated. In particular, certain aspects of video microscopy relating to frozen section evaluation have not been adequately assessed. We conducted a retrospective analysis of 48 excisional skin biopsy specimens encompassing a spectrum of benign and malignant lesions. To simulate an actual frozen section evaluation, only original frozen section slides were evaluated. Fields were selected and digitized (Roche Image Analysis System) by a pathology resident. Two sets of diagnoses were subsequently rendered by a surgical pathologist, the first set based on the digitized images and the second based on direct microscopic examination of the histological slides. The two sets of diagnoses were compared, and the concordance rates were as follows: malignant diagnoses, 100%; benign diagnoses, 100%; positive margins, 96%; negative margins, 99%. One (4%) of the 25 positive margins was indexed as negative by image analysis. Conversely, one (1%) of the 121 negative margins was indexed as positive by image analysis. In both of these cases, error was attributable to selection and digitization of an inappropriate field. We conclude that telepathology of static images is an accurate method of evaluating frozen sections of skin lesions. Potentially, this technology could be applied to the frozen section evaluation of other lesions as well. Static image analysis is, however, susceptible to errors induced by inappropriate field selection, emphasizing the need for trained and skillful personnel on both sides of the video camera.

Frozen Sections↗
PubMed2006

A novel culture system shows that stem cells can be grown in 3D and under physiologic pulsatile conditions for tissue engineering of vascular grafts.

BACKGROUND: Currently available vascular grafts have been limited by variable patency rates, material availability, and immunological rejection. The creation of a tissue-engineered vascular graft (TEVG) from autologous stem cells would potentially overcome these limitations. As a first step in creating a completely autologous TEVG, our objective was to develop a novel system for culturing undifferentiated mouse embryonic stem cells (mESC) in a three-dimensional (3D) configuration and under physiological pulsatile flow and pressure conditions. MATERIALS AND METHODS: A bioreactor was created to provide pulsatile conditions to a specially modified four-well Labtek Chamber-Slide culture system. Undifferentiated mESC were either suspended in a 3D Matrigel matrix or suspended only in cell-culture media within the culture system. Pulsatile conditions were applied to the suspended cells and visualized by video microscopy. RESULTS: Undifferentiated mESC were successfully embedded in a 3D Matrigel matrix and could withstand physiological pulsatile conditions. Video microscopy demonstrated that the mESC in the 3D matrix were constrained to the wells of the culture system, moved in unison with the applied flows, and were not washed downstream; this was in contrast to the mESC suspended in media alone. CONCLUSIONS: Undifferentiated mESC can be grown in 3D and under pulsatile conditions. We will use these results to study the effects of long-term pulsatile conditions on the differentiation of mESC into endothelial cells, smooth muscle cells, and fibroblast cells with the long-term goal of creating a completely autologous TEVG.

Animals↗
PubMed2006

Optical microscopy measurement of pair correlation functions.

We studied the pair correlation function g(r) of silica particles with a fluorescent core and a nonfluorescent shell which were confined between two glass plates by optical video microscopy. To investigate the possible role of optical artifacts due to overlapping particle images, we compared experiments, where, first, the whole particle (white image) and then, only the fluorescent core (fluorescent image) was used for determining particle positions. While under white-image conditions the observed g(r) exhibits a main peak at about 1.2 times the particle's diameter; under fluorescent image conditions the obtained g(r) resembles a short-ranged repulsive system where the main peak is close to contact. This discrepancy points towards artifacts of video microscopy, leading to erroneous g(r) and in turn to erroneous effective-pair potentials.

Journal Article↗
PubMed1991

Hepatic uptake of fluorescein, investigated by video fluorescence microscopy and digital image analysis.

We evaluated fluorescence microscopy combined with digitized image analysis for the investigation of fluorescein transport in the intact rat liver. The images of the surface of isolated rat livers which were perfused directly under a microscope were projected onto a silicone-intensified target camera, stored on a video tape and analyzed with a microcomputer equipped with an image digitizer. It was shown that, after correction of the day-to-day variability of the optical and electronical system, the increase in fluorescence intensity of the liver surface following fluorescein infusion depended linearly on the fluorescein concentration in the perfusion medium up to 1 mM. Since, under the conditions used, biliary secretion and metabolic influences were found to be insignificant the uptake mechanism is probably predominantly simple diffusion.

Animals↗
PubMed1994

A user's guide for avoiding errors in absorbance image cytometry: a review with original experimental observations.

The sources of errors which may occur when cytophotometric analysis is performed with video microscopy using a charged-coupled device (CCD) camera and image analysis are reviewed. The importance of these errors in practice has been tested, and ways of minimizing or avoiding them are described. Many of these sources of error are known from scanning and integrating cytophotometry; they include the use of white instead of monochromatic light, the distribution error, glare, diffraction, shading distortion, and inadequate depth of field. Sources of errors specifically linked with video microscopy or image analysis are highlighted as well; these errors include blooming, limited dynamic range of grey levels, non-linear responses of the camera, contrast transfer, photon noise, dark current, read-out noise, fixed scene noise and spatial calibration. Glare, contrast transfer, fixed scene noise, depth of field and spatial calibration seem to be the most serious sources of errors when measurements are not carried out correctly. We include a table summarizing all the errors discussed in this review and procedures for avoiding them. It can be concluded that if accurate calibration steps are performed and proper guidelines followed, image cytometry can be applied safely for quantifying amounts of chromophore per cell or per unit volume of tissue in sections, even when relatively simple and inexpensive instrumentation is being used.

Cytological Techniques↗
PubMed1994

[Blood flow in skeletal muscle during and after contraction: intravital microscopies studies].

Using an intravital video microscopy system, the red cell velocities, and the diameters of the capillaries and terminal arterioles were measured in rabbit tenuissimus muscle during and after contraction. Muscle contractions were produced by direct electric stimulation using square waves at 9 V of 0.1 msec duration and 2-6 Hz frequency. The red cell velocity gradually increased after stimulation. It was maximal after contraction in the capillaries, and was maximal during contraction in the terminal arterioles. When the stimulus frequency was high, then the maximal red cell velocity increased and returned to the level at rest more slowly than with low frequency, in both the capillaries and terminal arterioles. The proportion of capillaries with zero or very low velocities was reduced during and after contraction. There were few detectable changes in any capillary diameter during or after contraction. However the terminal arteriole diameter reduced during contraction and was dilated after contraction. These results suggest that the terminal arteriole might play an important part in the skeletal muscle blood flow control during and after contraction.

Animals↗