FREEZE SUBSTITUTION INTO LOWER TEMPERATURE SOLVENTS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 尽管对RF/FSF进入丙酮后的电磁场形态进行了许多出色的研究，但样品经常遭受冰晶损伤，这在更高分辨率的层析重建中可以检测到。虽然为了获得最佳效果，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过程可能会对冰层造成损害。由于丙酮的熔化温度为-94.9℃，冷冻替代需要在替代过程开始时将样品升温至-90℃，这明显高于标志着玻璃化冰和结晶冰之间的转变的-140℃[1]。对于混合蛋白质溶液，纯水的作用可能并不完全相同，因为细胞质的高溶质浓度可能会在一定程度上提高相变点，但可能无法达到丙酮的熔融温度。我们已经开始测试在较低温度(-140℃)下使用低熔点溶剂进行冷冻置换的优点，以努力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.