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.

项目概要 摘要 该项目的长期目标是改进冷冻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。冷冻电镜是观察亚细胞的一种有前途的方法 具有分子分辨率的原位组装。然而，冷冻电镜受到以下因素的阻碍： 冷冻玻璃化过程带来的不可重复性和样品厚度限制。 目前，玻璃化通常是通过将样品浸入低温流体中来实现的。这 即使在结构生物学中，低温浸入的过程仍然是众所周知的不可重复的 应用：通常需要进行许多低温浸入尝试才能获得高质量的非晶态 冰。在细胞生物学应用中，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 样品的尺寸达到微米级（<~10 μm），这限制了该技术的应用 稀疏的种子细胞。冷冻玻璃化过程将继续限制范围和产量 直到我们严格了解样品与冷冻剂相互作用的流体动力学 在低温骤降期间。一旦理解了这个过程，我们就可以对其进行设计以实现快速且 较厚样品的可重复冷却。优化冷冻玻璃化过程将解决 几个关键的技术障碍，包括：(i) 通过以下方式实现高通量样品处理： 提高样品制备的可重复性，(ii) 通过以下方式扩大冷冻电子断层扫描的范围： 增加适合低温浸入的样品的厚度，甚至（iii）实现时间- 通过以精确的时间间隔冷却样品来解决生物过程的纳米级成像 刺激后。 PI 组成了一个协作团队，具有独特的优势来解决这些问题 通过使用计算和实验方法相结合的技术障碍 通过 (1) 开发了解低温流动并扩展低温浸入的能力 模拟低温插入的计算工具，(2) 系统地探索设计空间和 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，并用它来（4）使用生物检验理论预测 样品。完成后，我们将进行理论驱动的实验来评估 最有前途的生物样品冷冻方法。新协议将增加 冷冻插入的再现性并将该技术扩展到更厚的样品，即 对于研究生物学相关的细胞组装和细胞间通讯是理想的。