Improving cryoEM and cryoFIB performance through amelioration of mechanical stress in vitreous ice.

项目摘要/摘要 玻璃化冷冻过程中引入的压力被认为可能是导致两个 实验观察到的方法学挑战：低温电子的分辨率损失 低温聚焦离子束球磨薄片的显微观察与磨损 微加工(CryoFIB)。CryoEM受到信息损失的限制，第一次 很少有电子撞击，而且越来越多的证据表明，电子束催化的弛豫 积聚的压力是起因。CryoFIB的一个限制是研磨的样品会破裂或 在铣削过程中由于应力而折断，但这种应力的来源尚未显示。 当样品通过急速冷冻制备时，水会膨胀，而 样本支持合同。破坏开放的氢键网络的添加剂 改变了水的热膨胀性。我们将使用体积调节辅料来 改变CryoEM和CryoFIB样品的热膨胀系数并消除 来自这个来源的机械应力。 冰的流变性表明，低温电磁网格的热退火将 让溶剂流动，缓解压力。我们将在没有预热的情况下预热低温EM网格 让它们去玻璃化以缓解压力。 因此，我们的目标是测试体积调节辅料或 热退火将解决束流诱导运动和 通过以下两个方面提高性能：1.提高CryoEM分辨率；2.提高 以低温FIB为单位的产量。

Project Summary/Abstract Stress introduced during vitrification has been proposed as a likely cause of two experimentally-observed methodological challenges: loss of resolution in cryo-electron microscopy (cryoEM) and attrition of lamellae milled in cryo-focused ion beam micromachining (cryoFIB). CryoEM is limited by a loss of information when the first few electrons strike, and mounting evidence points to beam-catalyzed relaxation of the built-up stress as the cause. A limitation of cryoFIB is that milled specimens break or snap during milling because of stress, but the source of this stress has not been shown. When specimens are prepared by plunge-freezing, water expands while the specimen support contracts. Additives that disrupt the open hydrogen-bonding network of water change its thermal expansivity. We will use volume-modulating excipients to change the thermal expansion coefficient of cryoEM and cryoFIB samples and eliminate mechanical stress from this source. Rheological properties of ice suggest that thermal annealing of cryoEM grids will allow solvent to flow and alleviate stress. We will warm cryoEM grids without devitrifying them to relieve stress. Therefore, our goal is to test whether volume-modulating excipients or thermal annealing will solve the problem of beam-induced motion and improve performance by 1. increasing resolution in cryoEM and 2. increasing yield in cryoFIB.