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.