Chandra S, Morrison G H, Wolcott C C
J Microsc. 1986 Oct;144(Pt 1):15-37. doi: 10.1111/j.1365-2818.1986.tb04670.x.
A freeze-fracture methodology was standardized for tissue culture cells to study intracellular distribution of diffusible elements with ion microscopy. Chinese hamster ovary (CHO) and normal rat kidney (NRK) cells grown on a silicon substrate were sandwiched using another smooth surface (silicon, glass, mica) in the presence of spacers and fast frozen in liquid nitrogen slush. The sandwich was fractured by prying the two halves apart under liquid nitrogen. This procedure produced large areas on the silicon substrate containing hundreds of cells grouped together and fractured at the apical cell surface. After freeze-drying, these cells revealed a subcellular distribution of Na, K, Ca, Mg, P, Cl and S with the approximately 0.5 micron lateral resolution of the ion microscope. Between the nuclei and the cytoplasm of cells, no major differences were observed for Na, K, Mg, P, Cl and S intensities. Calcium alone, however, exhibited a remarkable distribution. Calcium accumulated more in the cytoplasm than in the nuclei of cells. Even within the cytoplasm its distribution was heterogeneous, suggesting Ca binding sites. The fractured cells consistently exhibited high K-low Na intensities. The injured or dead cells were easily recognized among the healthy ones due to their abnormal ion composition. This simple freeze-fracture methodology allowed fracturing of cells without removing the cells from the substrate. In addition, it eliminated the need for washing the nutrient media away and cryo-sectioning before ion microanalysis. The methodology was successfully extended to 3T3 mouse fibroblast, PtK2 rat kangaroo and L5 rat myoblast cultures.
一种用于组织培养细胞的冷冻断裂方法被标准化，以利用离子显微镜研究可扩散元素的细胞内分布。将生长在硅基片上的中国仓鼠卵巢（CHO）细胞和正常大鼠肾（NRK）细胞，在间隔物存在的情况下，用另一个光滑表面（硅、玻璃、云母）夹住，并在液氮淤浆中快速冷冻。在液氮下将两半撬开使三明治断裂。该过程在硅基片上产生了大片区域，其中包含数百个聚集在一起并在细胞顶端表面断裂的细胞。冻干后，这些细胞显示出钠、钾、钙、镁、磷、氯和硫的亚细胞分布，离子显微镜的横向分辨率约为0.5微米。在细胞核和细胞质之间，钠、钾、镁、磷、氯和硫的强度没有观察到明显差异。然而，单独的钙表现出显著的分布。钙在细胞质中的积累比在细胞核中更多。即使在细胞质内，其分布也是不均匀的，表明存在钙结合位点。断裂的细胞始终表现出高钾低钠强度。由于其异常的离子组成，受损或死亡的细胞在健康细胞中很容易被识别。这种简单的冷冻断裂方法允许在不将细胞从基片上移除的情况下使细胞断裂。此外，它消除了在离子微分析之前冲洗掉营养培养基和冷冻切片的需要。该方法成功地扩展到了3T3小鼠成纤维细胞、PtK2大鼠袋鼠细胞和L5大鼠成肌细胞培养物。
