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

通过改善玻璃体冰的机械应力来提高冷冻电镜和冷冻聚焦离子束的性能。

• 批准号: 10475739
• 负责人: Jason T Kaelber
• 金额: $ 19.63万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10475739
• 关键词: 3-Dimensional; Attenuated; Benchmarking; Biological Assay; Biology; Cells; Contracts; Cryo-electron tomography; Cryoelectron Microscopy; Data Set; Dependovirus; Drug Design; Electron Beam; Electrons; Excipients; Freezing; Goals; Hydrogen Bonding; Ice; Image; Individual; Intervention; Ions; Joints; Maps; Mechanical Stress; Metals; Methodology; Monitor; Motion; Movement; Performance; Plant Roots; Problem Solving; Property; Proteins; Relaxation; Resolution; Sampling; Solvents; Source; Specimen; Stress; Structural Biologist; Structure; Support Contracts; System; Techniques; Temperature; Testing; Transmission Electron Microscopy; Water; cost; cryogenics; improved; innovation; lectures; method development; microscopic imaging; nanomechanics; particle; reconstruction; shear stress; solute; structural biology; technology research and development; theories; virtual

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.

项目总结/摘要 在玻璃化过程中引入的应力被认为是两种可能的原因。 实验观察到的方法学挑战：低温电子分辨率的损失 显微镜（cryoEM）和在低温聚焦离子束中研磨的薄片的磨损 微机械加工（cryoFIB）。CryoEM是有限的信息损失时，第一次 很少有电子罢工，越来越多的证据表明，束催化弛豫的 压力过大是原因cryoFIB的局限性是研磨的标本破裂或 在铣削过程中由于应力而折断，但未显示该应力的来源。 当标本是由暴跌冻结准备，水膨胀，而 支助合同样本。破坏开放氢键网络的添加剂 改变了水的热膨胀性。我们将使用体积调节赋形剂， 改变cryoEM和cryoFIB样本的热膨胀系数， 机械应力从这个来源。 冰的流变特性表明，低温EM网格的热退火将 允许溶剂流动并减轻应力。我们将加热冷冻电镜网格， 使其脱玻以缓解压力 因此，我们的目标是测试体积调节赋形剂或 热退火将解决束诱导运动的问题， 性能提升1.提高cryoEM的分辨率; 2.增加 cryoFIB产量。