FREEZE SUBSTITUTION INTO LOWER TEMPERATURE SOLVENTS

冷冻置换成低温溶剂

• 批准号: 8170850
• 负责人: MARY MORPHEW
• 金额: $ 3.74万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170850
• 关键词: Acetone; Computer Retrieval of Information on Scientific Projects Database; Crystal Formation; Cytoplasm; Freeze Substitution; Freezing; Funding; Grant; Ice; Institution; Morphology; Phase Transition; Process; Proteins; Research; Research Personnel; Resolution; Resources; Sampling; Solutions; Solvents; Source; Specimen; Temperature; Testing; United States National Institutes of Health; Water; cold temperature; melting; prevent; reconstruction; solute

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.

这个子项目是许多利用 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来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，但不一定是研究者所在的机构。 尽管对RF/FSF进入丙酮后的EM形态进行了许多出色的研究，但样本通常会遭受冰晶损坏，这在更高分辨率的断层重建中被检测到。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。