Merchant F A, Aggarwal S J, Diller K R, Bartels K A, Bovik A C
Biomedical Engineering Program, University of Texas, Austin 78712-1084.
J Microsc. 1993 Mar;169(Pt 3):329-38. doi: 10.1111/j.1365-2818.1993.tb03309.x.
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.
利用共聚焦显微镜进行连续光学切片技术，并结合离线数字图像分析，对冷冻保存后的大鼠胰岛中受损细胞的径向分布进行量化。该过程包括使用激光扫描共聚焦显微镜（LSCM）对冻融后的胰岛进行成像。通过吖啶橙/碘化丙啶（AO/PI）荧光染色可视化活细胞和受损细胞这两类细胞的三维（3-D）分布并进行分析。在准备冷冻保存时，将分离培养的大鼠胰岛在逐渐降低的温度下通过连续添加使其达到2 M浓度的二甲基亚砜（DMSO）。在-10℃下使胰岛悬液中形成冰核，然后在可编程大容量冷冻机中以1、3、10和30℃/分钟的冷却速率将单个样本冷冻至-70℃，随后储存在液氮中。快速解冻并连续稀释以去除DMSO后，将单个胰岛用AO/PI染色剂处理，以便在LSCM上成像。获得厚度为2 - 7微米的胰岛连续切片并进行处理以获得高对比度图像。分析算法包括模板掩蔽、灰度阈值处理、中值滤波和三维斑点着色。通过分离细胞并计算其与三维胰岛体积质心的距离来确定胰岛中受损细胞的径向分布。观察到与受损细胞的单个和/或聚集体相对应的斑点数量随着从胰岛中心向周边的距离逐渐增加。在处理的众多单个样本中，发现这种胰岛内细胞的冷冻诱导杀伤模式始终存在。
