FREEZE SUBSTITUTION INTO LOWER TEMPERATURE SOLVENTS

冷冻置换成低温溶剂

• 批准号: 8362552
• 负责人: MARY MORPHEW
• 金额: $ 2.13万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362552
• 关键词: Acetone; Cell physiology; Cellular Structures; Crystal Formation; Cytoplasm; Freeze Substitution; Freezing; Funding; Grant; Ice; Morphology; National Center for Research Resources; Phase Transition; Principal Investigator; Process; Proteins; Research; Research Infrastructure; Resolution; Resources; Sampling; Solutions; Solvents; Source; Specimen; Temperature; Testing; United States National Institutes of Health; Water; cold temperature; cost; melting; prevent; reconstruction; solute

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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 尽管在RF/FSF之后对EM形态进行了许多出色的研究，但样品通常会遭受冰晶损伤，这在较高的分辨率层析成像重建中被检测到。为了获得最佳结果，快速冷冻过程中样品的初始玻璃化是至关重要的第一步，但随后的RF/FSF工艺可能会施加其自身的冰损伤潜力。由于丙酮在-94.9 c时的熔化温度，冻结的替代需要在取代过程的主动性下将样品加热到-90°C，该替代过程明显高于-140O C，标志着玻璃化和结晶冰之间的过渡[1]。对于混合蛋白质溶液而言，纯净水的计算可能并不完全相同，因为高溶质的细胞质浓度可能会在某种程度上提高相转换点，但可能无法达到丙酮的熔化温度。我们已经开始测试在较低温度（-140¿C）开始冻结替代的优点，并结合了低融化溶液，以防止在冻结替代开始时冰晶形成。