FREEZE SUBSTITUTION INTO LOWER TEMPERATURE SOLVENTS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.