Giddings Thomas H, Morphew Mary K, McIntosh J Richard
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347.
Laboratory for 3D Electron Microscopy, University of Colorado, Boulder, Colorado 80309-0347.
Cold Spring Harb Protoc. 2017 Jan 3;2017(1):2017/1/pdb.prot091314. doi: 10.1101/pdb.prot091314.
Freezing samples while simultaneously subjecting them to a rapid increase in pressure, which inhibits ice crystal formation, is a reliable method for cryofixing fission yeast. The procedure consists simply of harvesting cells and loading them into a high-pressure freezer (HPF), and then operating the device. If equipment for high-pressure freezing is not available, fission yeast can be frozen by plunging a monolayer of cells into a liquid cryogen, usually ethane or propane. Unlike the HPF, where relatively large volumes of cells can be frozen in a single run, plunge freezing requires cells to be dispersed in a layer <20 µm thick. Unless frozen cells are to be imaged in the vitreous state, they must be fixed, dehydrated, and embedded for subsequent study by transmission electron microscopy; warming frozen cells without fixation badly damages cell structure. Fixation is best accomplished by freeze-substitution, a process in which frozen water is removed from samples by a water-miscible solvent that is liquid at a temperature low enough to prevent the cellular water from recrystallizing. Low concentrations of chemical fixatives and stains are generally added to this solvent such that they permeate the cells as the water is replaced. The activity of these additives is quite limited at the low temperatures required for minimizing ice crystal formation, but they are in the right place to react effectively as the cells warm up. Step-by-step protocols for HPF, plunge freezing, and freeze-substitution are provided here.
在对样品进行冷冻的同时使其承受快速的压力增加，这会抑制冰晶形成，是对裂殖酵母进行冷冻固定的可靠方法。该过程很简单，只需收获细胞并将其装入高压冷冻机（HPF），然后操作该设备即可。如果没有高压冷冻设备，裂殖酵母可以通过将单层细胞投入液态冷冻剂（通常是乙烷或丙烷）中进行冷冻。与高压冷冻机不同，在高压冷冻机中一次可以冷冻相对大量的细胞，而投入式冷冻要求细胞分散在厚度小于20 µm的层中。除非要对冷冻细胞进行玻璃态成像，否则必须对其进行固定、脱水和包埋，以便随后通过透射电子显微镜进行研究；未经固定就对冷冻细胞进行加热会严重破坏细胞结构。固定最好通过冷冻置换来完成，在这个过程中，冷冻水通过与水混溶的溶剂从样品中去除，该溶剂在足够低的温度下呈液态，以防止细胞内的水重新结晶。通常会向这种溶剂中添加低浓度的化学固定剂和染色剂，以便在水被置换时它们能渗透到细胞中。这些添加剂在将冰晶形成降至最低所需的低温下活性相当有限，但当细胞升温时，它们处于能有效反应的合适位置。此处提供了高压冷冻、投入式冷冻和冷冻置换的分步方案。
