Engineering fluid dynamics of cryo-plunging for improved vitrification

用于改善玻璃化的低温浸入的工程流体动力学

• 批准号: 10707442
• 负责人: Maxim Prigozhin
• 金额: $ 18.77万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-21 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10707442
• 关键词: Address; Biological; Biological Process; Cell Communication; Cell physiology; Cells; Cellular Structures; Cellular biology; Computer software; Convection; Coupled; Cryoelectron Microscopy; Cultured Cells; Custom; Eligibility Determination; Engineering; Feedback; Geometry; Glass; Goals; Ice; Image; In Situ; Investigation; Knowledge; Liquid substance; Methods; Modeling; Molecular; Molecular Structure; Monitor; Motion; Movement; Performance; Positioning Attribute; Preparation; Procedures; Process; Protocols documentation; Reproducibility; Resolution; Sampling; Series; Speed; System; Techniques; Temperature; Testing; Thick; Thinness; Time; Visualization; Water; Work; cellular imaging; computerized tools; cryogenics; design; electron tomography; experimental study; imaging modality; improved; instrument; manufacture; meter; millisecond; nanoscale; open source; sensor; simulation; solid state; structural biology; temporal measurement; theories; time interval

PROJECT SUMMARY ABSTRACT The long-term goal of this project is to improve cryo-vitrification sample preparation methods for cryo-electron microscopy (cryo-EM) and tomography (cryo-ET) in terms of their reproducibility and sample thickness limitations. Cryo-EM is a promising method for observing sub-cellular assemblies in situ with molecular resolution. However, cryo-EM is hampered by the irreproducibility and sample thickness limitations imposed by the cryo-vitrification process. Currently, vitrification is typically achieved by plunging the sample into a cryogenic fluid. This process of cryo-plunging remains notoriously irreproducible even in structural biology applications: many cryo-plunging attempts are typically required to get high-quality amorphous ice. In cell biology applications, the problem is exacerbated: the low thermal diffusivity of cells puts stringent requirements on the cooling rate in the vitrification process, limiting the thickness of the sample to the micron scale (<~10 μm), which restricts the application of this technique to sparsely seeded cells. The cryo-vitrification process will continue to limit the scope and throughput of cryo-EM until we rigorously understand the fluid dynamics of the sample-cryogen interaction during cryo-plunging. Once this process is understood, we can engineer it to achieve fast and reproducible cooling of thicker samples. Optimizing the cryo-vitrification process will address several critical technical barriers, including: (i) enabling high-throughput sample processing by increasing the reproducibility of sample preparation, (ii) expanding the scope of cryo-ET by increasing the thickness of samples eligible for cryo-plunging, and even (iii) achieving time- resolved nanoscale imaging of biological processes by cooling samples at precise time intervals after stimulation. The PIs form a collaborative team that is uniquely positioned to address these technical barriers by using a combination of computational and experimental methods to understand cryogenic flow and extend the capabilities of cryo-plunging by (1) developing computational tools to simulate cryo-plunging, (2) systematically exploring the design space and making testable predictions of system performance, (3) developing and validating a time-resolved temperature monitoring system, and using it to (4) test theoretical predictions using biological samples. Upon completion, we will have performed theory-driven experiments evaluating the most promising cryo-plunging protocols for biological samples. The new protocols will increase the reproducibility of cryo-plunging and extend this technique to thicker samples, which is desirable for investigation of biologically relevant cellular assemblies and cell-cell communication.

项目摘要 该项目的长期目标是改进低温玻璃化样品制备方法， 冷冻电子显微镜（cryo-EM）和断层扫描（cryo-ET）的再现性 和样品厚度限制。冷冻电镜是一种很有前途的亚细胞观察方法 分子分辨率的原位组装。然而，冷冻EM受到 不再现性和由低温玻璃化过程施加的样品厚度限制。 目前，玻璃化通常通过将样品浸入低温流体中来实现。这 即使在结构生物学中，冷冻过程也是众所周知的不可复制的 应用：通常需要许多低温插入尝试以获得高质量的非晶 冰在细胞生物学应用中，这个问题更加严重：细胞的低热扩散率 对玻璃化过程中的冷却速度提出了严格的要求，限制了厚度 样品的尺寸小于10 μm，这限制了该技术的应用， 种子稀疏的细胞低温玻璃化过程将继续限制范围和吞吐量 直到我们严格理解样品-冷冻剂相互作用的流体动力学 在低温下一旦理解了这个过程，我们就可以对其进行设计， 较厚样品的可重复冷却。优化低温玻璃化过程将解决 若干关键技术障碍，包括：（i）通过以下方式实现高通量样品处理： 增加样品制备的再现性，（ii）通过以下方法扩大冷冻ET的范围： 增加适合低温插入的样品的厚度，并且甚至（iii）实现时间- 通过以精确的时间间隔冷却样品的生物过程的分辨纳米级成像 刺激后。PI组成了一个协作团队，该团队具有独特的定位来解决这些问题 技术障碍，通过使用计算和实验方法相结合， 了解低温流动，并通过以下方式扩展低温柱塞的能力：（1）开发 计算工具，以模拟低温暴跌，（2）系统地探索设计空间， 对系统性能进行可测试的预测，（3）开发和验证时间分辨的 温度监测系统，并使用它来（4）测试理论预测，使用生物 样品完成后，我们将进行理论驱动的实验，评估 最有前途的生物样品冷冻方案。新协议将增加 低温插入的可重复性，并将该技术扩展到较厚的样品， 这对于研究生物学相关的细胞组装和细胞-细胞通信是理想的。