Grabenbauer Markus, Han Hong-Mei, Huebinger Jan
Institute for Anatomy and Cell Biology Germany, University of Heidelberg, Heidelberg, Germany.
Methods Mol Biol. 2014;1117:173-91. doi: 10.1007/978-1-62703-776-1_9.
High-pressure freeze fixation is the method of choice to arrest instantly all dynamic and physiological processes inside cells, tissues, and small organisms. Embedded in vitreous ice, such samples can be further processed by freeze substitution or directly analyzed in their fully hydrated state by cryo-electron microscopy of vitreous sections (CEMOVIS) to explore cellular ultrastructure as close as possible to the native state. Here, we describe the procedure of self-pressurized rapid freezing as fast, easy-to-use, and low-cost freeze fixation method, avoiding the usage of a high-pressure freezing (HPF) apparatus. Cells or small organisms are placed in capillary metal tubes, which are tightly closed and plunged directly into liquid ethane cooled by liquid nitrogen. In parts of the tube, crystalline ice is formed and builds up pressure sufficient for the liquid-glass transition of the remaining specimen. The quality of samples is equivalent to preparations by conventional HPF apparatus, allowing for high-resolution cryo-EM applications or for freeze substitution and plastic embedding.
高压冷冻固定是一种能瞬间停止细胞、组织和小型生物体内部所有动态和生理过程的首选方法。嵌入玻璃态冰中的此类样本可通过冷冻置换进一步处理，或通过玻璃态切片冷冻电子显微镜（CEMOVIS）在其完全水合状态下直接进行分析，以尽可能接近天然状态地探索细胞超微结构。在此，我们描述一种自加压快速冷冻方法，它是一种快速、易于使用且低成本的冷冻固定方法，无需使用高压冷冻（HPF）设备。将细胞或小型生物体置于毛细管金属管中，将其紧密封闭后直接投入由液氮冷却的液态乙烷中。在管的部分区域会形成结晶冰，并产生足以使剩余样本发生液 - 玻璃转变的压力。样本质量与使用传统HPF设备制备的样本相当，可用于高分辨率冷冻电镜应用或冷冻置换及塑料包埋。
