Wagner R C, Andrews S B
School of Life and Health Sciences, University of Delaware, Newark 19716.
J Electron Microsc Tech. 1991 Nov;19(3):276-90. doi: 10.1002/jemt.1060190304.
Cryofixation refers to the immobilization of tissue components by the rapid removal of heat from the specimen, so that the structure is interred and stabilized in a natural embedding medium, namely, frozen (amorphous or microcrystalline) tissue water. Cryofixation is now often used as a complement to the more traditional fixation methods, especially when the cell structure is delicate or dynamic and may be inaccurately preserved by the slow selective action of chemical fixatives. Vascular endothelial cells are specialized for transcellular transport and for the regulation of blood flow and composition. The dynamic and labile subcellular organization of these cells, presumably reflecting these functional specializations, makes them ideal candidates for cryofixation. Several different types of endothelial cells were directly frozen at temperatures below 20 degrees Kelvin by pressing them against a liquid-helium-cooled block. These samples were subsequently processed for structural analysis by freeze-substitution. Detailed rationales, designs, and protocols are described for both freezing and freeze-substitution. Electron micrographs of cryofixed arterial and venous capillaries (rete mirabile of the American eel), iliac vein (rabbit), and cultured endothelium from the iliac vein (human) reveal that the organization of the characteristic intracellular membrane system of endothelial vesicles is qualitatively similar to that seen in chemically fixed endothelium, especially with regard to the interconnection of clusters of individual vesicles to form elaborate networks. The luminal and abluminal networks are not in communication, at least not in static images. Quantitatively, however, most directly frozen endothelial cells have far fewer vesicular profiles than comparable glutaraldehyde-fixed cells. The differences can be explained by presuming that the rapid action of cryofixation (approximately 1 msec) gives a more accurate picture of the vesicular network because it captures the transient structure of labile or dynamic membranes.
冷冻固定是指通过迅速从标本中移除热量来固定组织成分，从而使结构在天然包埋介质即冷冻（无定形或微晶）组织水中得以保存和稳定。冷冻固定现在常被用作更传统固定方法的补充，特别是当细胞结构 delicate 或动态，可能会因化学固定剂的缓慢选择性作用而保存不准确时。血管内皮细胞专门用于跨细胞运输以及调节血流和成分。这些细胞动态且不稳定的亚细胞组织大概反映了这些功能特化，使其成为冷冻固定的理想候选对象。几种不同类型的内皮细胞通过压在液氦冷却的块上直接在低于 20 开尔文的温度下冷冻。随后对这些样品进行冷冻置换处理以进行结构分析。描述了冷冻和冷冻置换的详细原理、设计和方案。冷冻固定的动脉和静脉毛细血管（美洲鳗的神奇网）、髂静脉（兔）以及来自髂静脉的培养内皮（人）的电子显微镜照片显示，内皮小泡特征性细胞内膜系统的组织在质量上与化学固定的内皮相似，特别是在单个小泡簇相互连接形成复杂网络方面。管腔和管腔外网络不连通，至少在静态图像中不连通。然而，从数量上看，大多数直接冷冻的内皮细胞的小泡轮廓比可比的戊二醛固定细胞少得多。这些差异可以通过假设冷冻固定的快速作用（约 1 毫秒）给出更准确的小泡网络图像来解释，因为它捕捉了不稳定或动态膜的瞬时结构。
