Shanti J. Aggarwal

Correlation of HSP70 Expression and Cell Viability Following Thermal Stimulation of Bovine Aortic Endothelial Cells

Thermal preconditioning protocols for cardiac cells were identified which produce elevated HSP70 levels while maintaining high cell viability. Bovine aortic endothelial cells were heated with a water bath at temperatures ranging from 44 to 50 degrees C for periods of 1-30 min. Thermal stimulation protocols were determined which induce HSP70 expression levels ranging from 2.3 to 3.6 times the control while maintaining cell viabilities greater than 90%. An Arrhenius injury model fit to the cell damage data yielded values of A = 1.4 X 10(66) s(-1) and Ea = 4.1 X 10(5) J/mol. Knowledge of the injury parameters and HSP70 kinetics will enhance dosimetry guideline development for thermal stimulation of heat shock proteins expression in cardiac tissue.

Angiogenesis in cultured and cryopreserved pancreatic islet grafts.

BACKGROUND The ability of rat pancreatic islets to revascularize after transplantation was examined via in vitro and in vivo imaging of the microvasculature using laser scanning confocal microscopy (LSCM). METHODS Cultured or cryoprocessed islets were transplanted at the renal subcapsular site in rats. At various time intervals after transplantation, three-dimensional imaging of the graft was performed by LSCM. In vitro studies were conducted via microvascular corrosion casting of the grafted kidney in situations where it was difficult to obtain in vivo confocal data due to surgical complications. The vascular morphology of the islet grafts was evaluated quantitatively via digital image analysis algorithms to determine the morphology of the neovascular ingrowth and the rate of revascularization. RESULTS In cultured islet grafts, the initiation of angiogenesis was observed within 1 week, characterized by the presence of capillary sprouts, tortuous vessels, and blood vessels with blind ends. The revascularization of the graft was typically completed within 2 weeks and could be distinguished as a network of completely perfused blood vessels consisting of intertwining capillaries, with surrounding arterioles and venules. The angiogenesis process in cryopreserved islet grafts required a longer time period to initiate (approximately 2 weeks), and the revascularization was completed in 1 week after the initiation. CONCLUSIONS These results successfully demonstrate the potential of the described in vivo and in vitro LSCM techniques to measure the angiogenesis process in pancreatic islet grafts.

Shape description of biological objects via stereo light microscopy

Computational stereo vision techniques are applied to the analysis of the three-dimensional (3-D) shape of biological specimens imaged through a stereo light microscope. 3-D shape descriptions are derived by integrating binocular and monocular measurements. Microscopic biological objects viewed through a light microscope generally have ill-defined boundaries in shape and reflectance and often exhibit transparency. In addition, the limited illumination available in the light microscope often makes it difficult to obtain images of sufficiently high contrast. In the approach presented, a new matching algorithm is introduced using intensity gradient information to solve the problem. Shape descriptions are computed by exploiting a number of additional processing steps. Two different classes of shapes are considered, which lead to two distinct approaches for 3-D analysis: solid objects and vascular networks. The first class of objects is described using a visible surface representation, while the second is expressed in terms of a set of space curves. The efficacy of each approach is demonstrated using microscopic-scale solid and microvascular specimens. >

Cryomicroscopic determination of the membrane osmotic properties of human monocytes at subfreezing temperatures.

Monocytes were isolated from fresh whole human blood and resuspended in Hanks balanced salt solution; a portion of the cells was mixed with an equal volume of 2M dimethyl sulfoxide (DMSO) to form a 1 M solution. Microliter volumes of cell suspension were placed directly onto a computer-controlled cryostage and cooled to a predetermined subzero temperature. Ice was nucleated in the extracellular medium and a continuous video record was made of the subsequent osmotically induced volume changes of individual cells owing to exposure to the concentrated extracellular solutes. Selected micrographs emphasizing the initial transient data were digitized for computer analysis with an interactive boundary tracing algorithm to determine metric parameters of specific cells, and apparent volume changes were measured as a function of elapsed time after nucleation. The Kedem-Katchalsky-coupled transport equations were fit to the data using a network thermodynamic model implemented on a microcomputer to determine values for the permeability properties Lp, omega, and sigma. Experiments were performed over the temperature range from -7 degrees to -10 degrees C. Cells pre-equilibrated with DMSO had a lower Lp and a higher activation energy, delta E, than without additive, although the statistical significance of the difference could not be substantiated. It was found that the movement of DMSO across the plasma membrane in response to extracellular freezing was apparently so much smaller than the water flux that values for omega and sigma could not be determined from the data base.

In-vivo analysis of angiogenesis and revascularization of transplanted pancreatic islets using confocal microscopy

A technique to measure angiogenesis and revascularization in pancreatic islets transplanted at the renal subcapsular site in the rat has been developed. In‐vivo imaging of the microcirculation of transplanted pancreatic islets was conducted using a confocal scanning laser microscope (CSLM) to achieve optical sectioning through the graft in order to perform a computer reconstruction of the three‐dimensional neovascular morphology. Individual islets were harvested by enzymatic digestion of excised pancreas from Fischer 344 rats. Isolated islets were cultured for 24 h, and approximately 300–350 islets were transplanted at the renal subcapsular site of the left kidney in an anaesthetized rat. Six to 14 days post‐transplantation, the animal was anaesthetized and prepared for in‐vivo imaging of the microvasculature on a Zeiss LSM‐10. Optical contrast of the microvasculature was enhanced by the administration of fluorescein‐labelled dextran into the circulating blood. The transplant site was identified and serial sections were obtained through the vascular bed at varying z‐intervals. Complementary fluorescence video images were also obtained via a silicon intensifier tube camera mounted on the CSLM. At completion of the imaging procedure, the kidney was returned into the body cavity, the area was sutured and the animal was allowed to recuperate for subsequent examinations. Image processing algorithms, such as grey‐level thresholding, median filtering, skeletonization and template matching, were applied to compute the vessel density and diameters and extrapolated to measure 3‐D vessel lengths and the tortousity index of the neovasculature.

A CHARACTERIZATION OF THE FLUORESCENT PROPERTIES OF CIRCULATING HUMAN EOSINOPHILS

Abstract— This paper presents a characterization of the fluorescence properties of human eosinophils isolated from peripheral blood of normal donors over a wide range of excitation and emission wavelengths. Circulating eosinophils possess three fluorescence excitation emission maxima: one at 280 nm excitation, 330 nm emission, attributable to tryptophan fluorescence, and currently unassigned peaks at 360 nm excitation, 440 nm emission and 380 nm excitation, 415 nm emission. Fluorescence microscopy studies show that the Huorescence of eosinophils may be site dependent; specifically, when observed at 365 nm excitation, circulating eosinophil Huorescence appears blue‐violet, while the fluorescence of tissue‐dwelling eosinophils appears amber‐gold. These results should be considered in developing an optical biopsy technique to identify eosinophils in human tissue.

The reconstruction of dynamic 3D structure of biological objects using stereo microscope images

Abstract.In this paper, we address the analysis of 3D shape and shape change in non-rigid biological objects imaged via a stereo light microscope. We propose an integrated approach for the reconstruction of 3D structure and the motion analysis for images in which only a few informative features are available. The key components of this framework are: 1) image registration using a correlation-based approach, 2) region-of-interest extraction using motion-based segmentation, and 3) stereo and motion analysis using a cooperative spatial and temporal matching process. We describe these three stages of processing and illustrate the efficacy of the proposed approach using real images of a live frogs ventricle. The reconstructed dynamic 3D structure of the ventricle is demonstrated in our experimental results, and it agrees qualitatively with the observed images of the ventricle.

Digital reconstruction of three-dimensional serially sectioned optical images

The nature of out-of-focus noise is analyzed and linear filtering approaches are demonstrated for minimizing its effect. The analysis is based on concepts of geometrical optics, and takes into account the passage of light through the object. The object is assumed to be nondiffractive and to have low contrast. These assumptions, although not usually met in practice, are not overly restrictive. Experience shows that even dark, diffractive objects show substantial improvement after filtering. Sample images of a pollen grain and a rat pancreas islet are used to demonstrate the efficacy of the technique. >

Hydraulic permeability and activation energy of human keratinocytes at subzero temperatures

Studies on isolated human keratinocytes provide a model for design of optimal freeze-thaw protocols for skin cryopreservation and banking. Nucleated keratinocytes from the basal layer of split thickness human cadaveric skin were separated by a combined trypsin and DNAse digestion and suspended in Dulbeccos minimal essential medium with fetal calf serum. A small volume of suspension was frozen on a microprocessor controlled cryostage. Extracellular ice was nucleated at predetermined subzero temperatures, and the temperature was held constant for the duration of the experiment. The osmotic response of the cells to the formation of extracellular ice was recorded on 35-mm photographic film. Selected serial frames were digitized for automated computer evaluation of metric parameters of specific cells. Changes in the apparent cell volume were quantified over a period of several minutes to obtain dehydration curves associated with exposure to concentrated extracellular electrolytes. The Kedem-Katchalsky coupled flow transport model was statistically fit to the data using a personal computer. Values for the permeability coefficients were adjusted to optimize the correlation between the theory and the data. An activation energy of 44.8 kJ/mol and a water permeability of 0.035 micron (atm.min) at 0 degrees C were derived from the data measured over a temperature range from -2 to -9 degrees C.

Improvement in the resolution of three‐dimensional data sets collected using optical serial sectioning

In this paper an approach for improving the quality of 3‐D microscopic images obtained through optical serial sectioning is described and implemented. A serially sectioned image is composed of a sequence of 2‐D images obtained by incrementing the focusing plane of the microscope through the specimen of interest; ideally, the image obtained at each focusing plane should be in focus, and should contain information lying only within that plane. In practice, however, the images obtained contain redundant information from neighbouring focusing planes and are blurred by a three‐dimensional low‐pass distortion. These degradations are a consequence of the limited aperture of any optical system; using principles of geometric optics and allowing for the passage of light through the specimen, we are able to demonstrate that the microscope distortion can be described as a linear system, if the absorption of the specimen is assumed to be linear and non‐diffractive. The transfer function of the microscope is found to zero a biconic region of 3‐D spatial frequencies orientated along the optical axis; a closed‐form expression is derived for the low‐pass transfer function of the microscope outside the region of missing frequencies. The planar resolution of the serial sections can be greatly improved by convolving the image obtained with the inverse of the low‐pass distortion function, although the missing cone of frequencies is not recoverable. The reconstruction technique is demonstrated using both simulated images, to demonstrate more clearly the effects of the distortion and the accuracy of the subsequent reconstruction, and actual experiments with a pollen grain and a stained preparation of human cerebellum tissue.