Algae permeability to Me(2)SO from -3 to 23 degrees C.

Biphasic transport of water and dimethyl sulfoxide (Me(2)SO), a common cryoprotective agent (CPA), in algal cells was induced and measured on a cryoperfusion stage. A two-step experimental protocol provided data for the volumetric response of Chlorococcum (C.) texanum to impermeable and permeable solutes. First, the cells were exposed to a 500-mOsm sucrose solution, causing immediate shrinkage of the cell to a minimum equilibrium volume. Then an isoosmotic 200-mOsm/300-mOsm CPA/sucrose solution was introduced to the cells, resulting in increased cell volume to a new equilibrium state. Experiments were conducted at temperatures between -3 and 23 degrees C. Cell volumes were measured off-line by computer analysis of video images. A network thermodynamic model was fit to the transient volume data to determine permeabilities of C. texanum to water and Me(2)SO over the full temperature range, and results were calculated with two numeric methods. Biphasic transport was found to be slower at colder temperatures, with water entering the cell faster than Me(2)SO. Experimental results were also compared with data from similar experiments using methanol (MeOH) as the CPA. MeOH influx was calculated to be a magnitude larger than that of water. Additionally, MeOH permeability was at least three orders of magnitude greater than Me(2)SO permeability, and the difference in these solute permeabilities increased as temperature decreased.

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.

Viability analysis of cryopreserved rat pancreatic islets using laser scanning confocal microscopy.

We have developed a digital image analysis technique to assay the viability of frozen-thawed pancreatic islets by using laser scanning confocal microscopy (LSCM) in conjunction with double fluorescent staining [acridine orange/propidium iodide (AO/PI)]. Freshly isolated rat pancreatic islets were cultured for 18-24 h and then brought to a 2 M concentration of dimethyl sulfoxide (Me2SO) by serial addition at decreasing temperatures. Ice was nucleated in the islet suspension at a defined temperature (-10 degrees C), followed by a controlled period for equilibration and then cooling in a programmable bulk freezer at rates of 0.3, 1, 3, 10 and 30 degrees C/min to -70 degrees C. Samples were then stored in liquid nitrogen. Subsequent to rapid thawing and serial dilution with sucrose solution to remove Me2SO, AO/PI-stained individual islets were prepared for imaging on the LSCM. A series of optical sections through individual stained islets were obtained and processed to obtain high-contrast images at two different wavelengths; 488 nm and 514 nm for viable and damaged tissue, respectively. Image analysis algorithms consisted of template masking, generation of histograms of the pixel intensity profile, and gray level thresholding to obtain binary images. The total percentages of both types of tissue in the islet were computed by summing the two populations in each serial section. The spatial distributions of viable and damaged tissue were calculated from the three-dimensional (3-D) data base for both cultured (control) and cryopreserved islets. The 3-D spatial distributions of damaged and viable tissue in the islets were computed by determining the normalized distance of each viable/damaged voxel from the centroid of the islet volume to a mathematically estimated 3-D superquadric surface used to estimate the outer boundary of the islet. Further, each isolated damaged cell was identified and its volume determined. These studies indicate that cryopreserved islets exhibit shape distortion and a decrease in the numerical density of cells in comparison to unfrozen controls. Maximal survival was observed at the slower cooling rates. Accordingly, damage was found to occur throughout the 3-D islet volume in distinct spatial distributions for islets frozen at the slower and the faster cooling rates. Further, it was found that the volume of the majority of damaged cells identified was consistent with that of cells ranging in diameter from 5 to 9 micrometers.

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.

Local macromolecular extravasation in thermal burns quantified by fluorescent video microscopy and computer vision.

A dorsal skin flap chamber model was developed for analysis of the microvascular response to moderate intensity local thermal burns. Fluorescein isothiocyanate tagged 70,000 d dextran was introduced to visualize the extravasation and interstitial transport of macromolecules at the burn site. Contact burns 0.5 cm in diameter were affected by touching a thermostated metal rod onto the exposed epidermal side of the chamber preparation. All burns were of 5-second duration at temperatures between 55 degrees C and 70 degrees C. Postburn leakage of the fluorescein-labeled probed was monitored at numerous sites in the preparation on a fluorescent microscope equipped with a low-light-level intensified silicon intensified target video camera and recorded on tape for subsequent quantitative analysis. Selected scenes were digitized and subjected to a sequence of computer-image processing operations to extract quantitative information about the concentration distribution and net accumulation of dextran in the interstitial space as a function of postburn time. A diffusion model based on cylindrical geometry was fit to the concentration profile data at each site analyzed, and an apparent diffusion coefficient describing the interstitial transport process was determined. The interstitial transport increased with burn temperature up to a threshold of 70 degrees C, where other factors resulted in significant reduction in the loss of fluorescent macromolecule from the vasculature.

Burn-induced alterations in vasoactive function of the peripheral cutaneous microcirculation.

Alterations in the cutaneous vasomotion function caused by scald burns to a large area on the flank were observed and quantified in a dorsal skin flap window with a hamster model. Subsequent to chronic implantation of the window chamber to provide direct microscopic observation of blood flow for an entire thickness of skin, control measurements of vasoactivity in a defined network of arterioles were taken by means of a digital video image splitter over a period of several days. A 100 degrees C, 5-second water scald was then effected over 17% to 55% of the total body surface area, and the vasoactivity was remeasured in the targeted set of sites at serial time intervals for 2 to 4 days. Data were acquired directly into a computer and analyzed for both the magnitude and time pattern of diameter fluctuations during 3-minute observation periods. Average and standard deviations of diameters were computed, and the Prony power spectral analysis method was applied to identify the presence and strength of fluctuation frequencies. In general, vasomotion was suppressed for several hours subsequent to the burn, and the activity was dominated by much slower contractions than control. No direction of vessel diameter changes prevailed uniformly throughout the peripheral microcirculation. Large alternate constrictions and dilations were also observed for some arteriolar and venular components, but others exhibited no significant change at all. The incidence and magnitude of peripheral microvascular response was proportional to the size of the injured area.

The analysis of biological shape changes from multidimensional dynamic images.

A technique for modeling shape changes in a time series of biological images of arbitrary dimension is described. The technique consists of first segmenting the image to locate the specimen, and then parametrizing the specimen in the initial image with an orthogonal material coordinate system. The deformation of the material coordinate system caused by the changing shape of the specimen is then solved for by minimizing an energy functional. The energy functional is a linear combination of a brightness continuity term and a shape change term. A parameter lambda, weights the brightness continuity against the shape change smoothness. The best value to use for lambda is chosen as the value that minimizes the mean square error between the image derived from the calculated shape change parameters and the corresponding actual image. A two-dimensional implementation by finite differences is given. Results from both two-dimensional confocal images, and two-dimensional synthetic images are presented. Our early work on a three-dimensional implementation is given.

Three-dimensional distribution of damaged cells in cryopreserved pancreatic islets as determined by laser scanning confocal microscopy.

The technique of serial optical sectioning by confocal microscopy, in conjunction with off-line digital image analysis, was used to quantify the radial distribution of damaged cells in rat pancreatic islets following cryopreservation. The process consists of imaging frozen-thawed islets of Langerhans using laser scanning confocal microscopy (LSCM). The three-dimensional (3-D) distribution and analysis of the two populations of viable and damaged cells was visualized via acridine orange/propidium iodide (AO/PI) fluorescent staining. In preparation for cryopreservation, isolated and cultured rat pancreatic islets were brought to a 2 M concentration of dimethyl sulphoxide (DMSO) by serial addition at decreasing temperatures. Ice was nucleated in the islet suspension at -10 degrees C, and individual specimens were frozen to -70 degrees C at cooling rates of 1, 3, 10 and 30 degrees C/min in a programmable bulk freezer and subsequently stored in liquid nitrogen. After rapid thawing and serial dilution to remove DMSO, individual islets were prepared with AO/PI stains for imaging on the LSCM. Serial sections of the islets, 2-7 microns in thickness, were obtained and processed to obtain high-contrast images. Analysis algorithms consisted of template masking, grey-level thresholding, median filtering and 3-D blob colouring. The radial distribution of damaged cells in the islets was determined by isolating the cell and computing its distance from the centroid of the 3-D islet volume. An increase in the number of blobs corresponding to single and/or aggregates of damaged cells was observed progressively with distance from the centre towards the periphery of the islet. This pattern of freeze-induced killing of cells within the islet was found to occur consistently in the numerous individual specimens processed.

Analysis of volumetric changes in rat pancreatic islets under osmotic stress using laser scanning confocal microscopy.

Analysis of the volumetric changes in rat pancreatic islets undergoing shrinkage and/or swelling due to osmotic stress is essential for understanding the mechanism of mass transport between cells and their environment and for optimizing cryopreservation protocols. Addition and removal of cryoprotective additives is an integral component of all cryopreservation processes. We have used laser scanning confocal microscopy (LSCM) of acridine orange/propidium iodide (AO/PI) stained Islets of Langerhans to analyze the effects of osmotic stress induced by exposure to varying concentrations of the cryoprotectant dimethyl sulfoxide (DMSO), on the islet volume at two temperatures 23 degrees C and 15 degrees C. Experiments were conducted by mounting a single islet onto a unique freeze-thaw-perfusion stage on which the system temperature and the chemical composition of the solutions can be precisely controlled. The bathing medium of the islet was rapidly changed from isotonic saline to the desired DMSO osmolality to produce a defined osmotic stress, and the islet was imaged simultaneously using 488 nm argon laser. Three to seven serial sections were obtained through each islet at increments varying between 15 microns and 20 microns. The three-dimensional (3-D) image was segmented into islet and non-islet regions using a combination of median filtering, gray level thresholding and region labeling, and the islet volume was computed by counting voxels. Further, a special analysis algorithm was applied to identify shape changes both locally and globally throughout the islet volume.

Selective laser sintering for the creation of solid models from 3D microscopic images.

An exciting application of using Selective Laser Sintering (SLS) to produce solid models of microscopic specimens imaged with a laser scanning confocal microscope is presented. The SLS model, fabricated by sintering together successive layers of a fine powder with a computer controlled scanning laser, is a true 3-D magnification of the microscopic sample. The 3-D models produced are accurate representations of the data and can be handled and manipulated to evaluate surface details and morphology. The models give an extremely powerful method for 3-D data visualization and tactilization. Three dimensional digital image processing is performed on microscopic images to prepare the data for the selective laser sintering process. The image processing techniques for preparing 3-D images of both translucent and opaque specimens for laser sintering are presented. For translucent specimens, the image processing removes noise and "fills in" inclusions and cavities within the specimen data in order to produce a structurally sound model. When imaging an opaque specimen an image of the upper surface alone is acquired. Image processing is used to remove noise and to Fill the volume under the specimen surface. Sample models of a dandelion (Taraxicum officinale Compositae) pollen grain and the surface of a U.S. penny are presented. These models present examples of both the translucent and opaque data types.

The effects of epinephrine, ibuprofen, and tetrachlorodecaoxide on cutaneous microcirculation in thermally injured hamsters.

Fluctuations in the diameter of selected arterioles in the cutaneous microcirculation of Syrian golden hamster dorsal skin flap chambers, which ranged in size from 10 to 70 microns at different branching order sites, were measured before burn, at the same sites after burn and after injection of the drug. Three different drugs epinephrine (administered intravenously), ibuprofen (administered intravenously), and tetrachlorodecaoxide (administered intravenously and topically) were evaluated. Results show that the response to thermal injury in the control group involved extensive vasodilation in the arterioles, prolonged flow irregularities including flow obstructions and stasis, and a decrease in the level of vasoactivity of the microvessels. In two treatment groups, the ibuprofen and tetrachlorodecaoxide groups, significant improvement as indicated by reduced vasodilation and edema and improved microcirculatory blood flow after injury were observed. Further testing of tetrachlorodecaoxide as a topically applied wound dressing is indicated.

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.

Simultaneous multiple site arteriolar vasomotion measurement using digital image analysis.

An automatic digital image processing technique for simultaneous vasomotion analysis in peripheral microcirculation at multiple sites and in real time is presented. The algorithm utilizes either fluorescent or bright field micro-images of the vasculature as input. The video images are digitized and analyzed on-line by an IBM RT PC, using digital filtering and edge detection. The sampling frequency is higher than 5 Hz when only one site is tracked and decreases as the number of sites is incremented. Performance of the algorithm was tested for a hamster cutaneous microcirculation model.

The effects of burn injury on vasoactivity in hamster peripheral microcirculation.

The effects of mild thermal shock on the vasoactivity of microvessels were studied in the hamster skin flap window preparations. Diameter fluctuations in arterioles and venules, varying in size from 10 to 50 microns and at different branching order sites, were measured prior and subsequent to a local surface skin burn. The experimental vasomotion data were characterized in terms of mean, standard deviation, skewness, and kurtosis, and by the Prony spectral line estimator (PSLE), fast Fourier transform (FFT), and auto regression (AR) methods of spectral analysis. Following a mild burn the A1 (37-50 microns), A2 (30-50 microns), and A3 (25-41 microns) arterioles relaxed to a larger diameter by an average of 58, 20, and 13%, respectively. Dispersion statistics postburn showed a skewness close to normal while the kurtosis became more negative, indicating that the transient diameter curves were flatter. Both FFT and PSLE analyses indicate less energy in the signals postburn accompanied by a shift toward lower frequencies with decreased amplitude. In addition, there was a loss of certain frequencies from the spectrum and an increase in the interval for rhythmic activity. FFT analysis gave an idea of the trend while PSLE analysis was found to be highly unstable and dependent on the window size. AR results indicate that the process is stable and definite trends exist in the data, however, the data are not purely periodic.

3-D model of vascular network in rat skin obtained by stereo vision techniques.

The quantitative analysis of the depth of injury, penetration of therapeutic agents in tissues, and the regeneration of vascular patency after a graded degree of thermal injury requires a knowledge of the shape and spatial configuration of the vascular networks in the tissue. We have applied computational stereo vision techniques to describe the 3-D configuration of microvessels in full thickness rat skin vascular casts produced by perfusion of Yellow Microfil latex solution through the aorta. The principal concern is to describe the 3-D structure of vascular networks using a set of 3-D space curves. This representation is computed by integrating monocular and binocular processing; the 2-D curve representation of blood vessels computed through monocular analysis is integrated with disparity data to yield a space curve representation for each vessel. A connection diagram is also computed to indicate the connections existing among the computed space curve representations.

Laser-irradiation-induced relaxation of blood vessels in vivo.

The response of blood vessels to laser irradiation in vivo was studied in the dorsal skin flap glass window chamber model of hamsters. The vasodilatory response of venules was critically dependent on the wavelength of irradiating laser. Relaxation was not produced in arterioles, although it was tried repeatedly. Vessels were irradiated with the 514.5 nm single line argon laser with irradiances from 1 to 10 W/cm2 on a 1.2 mm-diameter spot. Irradiation of venules with 2.2 W/cm2 and 4.25 W/cm2 produced reversible relaxation. Venules relaxed initially and after the interruption of irradiation returned to their original diameter. At higher irradiances (8.5 W/cm2) an irreversible relaxation was observed. At irradiances of 10 W/cm2 and above initial relaxation was accompanied with constriction, focal coaguli, and hemostasis. Irradiation with the argon-pumped dye laser at 595 nm did not produce any significant relaxation.

Fluorescence digital microscopy of interstitial macromolecular diffusion in burn injury.

Computer vision techniques implemented on an IBM PC/AT have been applied to the study of microvascular permeability and interstitial diffusion in dorsal skin flap chamber preparations of hamsters. Experimental data was obtained for the leakage of fluorescent labelled dextran (70,000 daltons) after a precisely controlled mild degree of localized thermal trauma and compared with control data acquired prior to burn injury. Computer vision analysis techniques were applied to convert the fluorescent images into two-dimensional concentration maps. Interstitial diffusion coefficient values were computed from measured extravascular concentration profiles around a vessel of interest, assuming cylindrical or rectangular geometry, and optimally fitting a diffusion model to the data. An increase in the apparent diffusivity after mild thermal trauma was observed. Novel techniques were applied to solve hardware problems related to data acquisition and analysis, and a new library of software was developed to handle specific image processing requirements.

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 Dulbecco's 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.

Computer automated cell size and shape analysis in cryomicroscopy.

Computer vision techniques have been developed for quantitative analysis of size and shape changes in cells frozen on a cryomicroscope. The analysis is based on implementation of standard serial edge detection algorithms in conjunction with a shape transform to isolate individual cells in complex scenes which may include adjacent and overlying ice crystals. In the present study the sensitivity of the automated analysis procedure is evaluated for images obtained by various microscope optical systems for progressive degrees of subject blurring by defocusing. Size measurements in calibration trials for freezing latex spheres with extracellular ice in the field of view were least sensitive for bright field images, although the most consistent data was obtained by differential interference contrast microscopy. In all cases phase contrast images produced the least accurate data. An example analysis is presented for the freezing of pancreas beta-cells.