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Three‐dimensional distribution of damaged cells in cryopreserved pancreatic islets as determined by laser scanning confocal microscopy
Author(s) -
MERCHANT F. A.,
AGGARWAL S. J.,
DILLER K. R.,
BARTELS K. A.,
BOVIK A. C.
Publication year - 1993
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1993.tb03309.x
Subject(s) - confocal , islet , cryopreservation , propidium iodide , acridine orange , confocal microscopy , pancreatic islets , microscopy , confocal laser scanning microscopy , chemistry , fluorescence microscope , staining , materials science , biomedical engineering , biophysics , biology , fluorescence , optics , microbiology and biotechnology , biochemistry , insulin , physics , medicine , embryo , apoptosis , genetics , programmed cell death , endocrinology
SUMMARY 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°C, and individual specimens were frozen to −70°C at cooling rates of 1, 3, 10 and 30°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 μm 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.