Confocal microscopy and 3-D distribution of dead cells in cryopreserved pancreatic islets

摘要

Our laboratory is involved in studies of changes in shape and size of biological specimens under osmotic stress at ambient and sub-zero temperatures. This paper describes confocal microscopy, image processing and analysis of 3-D distribution of cells in acridine orange/propidium iodide (AO/PI) fluorescent stained frozen-thawed islet of Langerhans. Isolated and cultured rat pancreatic islets were frozen and thawed in 2 M dimethylsulfoxide and examined under a Zeiss laser scanning confocal microscope. Two $mu@m to five $mu@m serial sections of the islets were obtained and processed to obtain high contrast images which were later processed in two steps. The first step consisted of the isolation of the region of interest by template masking followed by grey level thresholding to obtain a binary image. Three- dimensional blob coloring algorithm was applied and the number of voxels in each region and the number of regions were counted. The volumetric distribution of the dead cells in the islets was computed by calculating the distance from the center of each blob to the centroid of the 3-D image. An increase in the number of blobs moving from the center toward the periphery of the islet was observed indicating that the freeze damage was more concentrated in the outer edges of the islet.