FREEZE SUBSTITUTION INTO LOWER TEMPERATURE SOLVENTS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 尽管RF/FSF进入丙酮后的EM形态的许多优秀的研究，样品往往遭受冰晶损伤，这是在更高分辨率的层析重建检测。虽然为了获得最佳结果，在快速冷冻过程中样品的初始玻璃化是关键的第一步，但随后的RF/FSF过程可能会造成其自身的冰损伤。由于丙酮的熔化温度为-94.9 ° C，冷冻置换需要在置换过程开始时将样品加热到-90 ° C，这明显高于标志着玻璃化和结晶冰之间转变的-140 ° C [1]。 纯水的重要性对于混合蛋白质溶液可能不完全相同，因为细胞质的高溶质浓度可能在一定程度上提高相变点，但可能不会达到丙酮的熔化温度。我们已经开始测试在较低温度（-140 ° C）下使用低熔点溶剂组合开始冷冻置换的优点，以防止在冷冻置换开始时形成冰晶。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Despite the many excellent studies of EM morphology after RF/FSF into acetone, samples often suffer from ice crystal damage, which is detected in the higher resolution tomographic reconstructions. While for best results the initial vitrification of samples during rapid freezing is a crucial first step, the subsequent RF/FSF process may impose its own potential for ice damage. Due to the melting temperature of acetone at -94.9¿C, freeze substitution requires warming the specimen to -90¿C at the initiation of the substitution process which is significantly higher than the -140o C that marks the transition between vitrified and crystalline ice [1]. What counts for pure water may not be exactly the same for a mixed protein solution as the high solute concentration of the cytoplasm may raise the phase-transition point to some extent, but likely not to reach the melting temperature of acetone. We have begun to test the merit of starting freeze substitution at lower temperatures (-140¿C) with a combination of low-melting solvents in an effort to prevent ice crystal formation at the onset of freeze substitution.