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: 10430822
• 负责人: Maxim Prigozhin
• 金额: $ 22.55万
• 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/10430822
• 关键词: Address; Biological; Biological Process; Cell Communication; Cell physiology; Cells; Cellular Structures; Cellular biology; Computer software; Convection; Coupled; Cryo-electron tomography; Cryoelectron Microscopy; Custom; Diffuse; Engineering; Feedback; Geometry; 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; Water; Work; base; cellular imaging; computerized tools; cryogenics; crystallinity; design; experimental study; imaging modality; improved; instrument; millisecond; nanoscale; open source; sensor; simulation; solid state; structural biology; temporal measurement; theories; time interval; tomography

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.

项目摘要摘要 该项目的长期目标是改善冷冻剂量样本准备方法 冷冻电子显微镜（冷冻-EM）和断层扫描（Cryo-ET）就其可重复性而言 和样品厚度限制。 Cryo-Em是观察亚细胞的有前途的方法 分子分辨率的原位组件。但是，冷冻EM受到了 不可重复的性能和样本厚度限制由冷冻剂量化过程施加的。 目前，通常通过将样品浸入低温流体中来实现玻璃化。这 即使在结构生物学中，臭名昭著 申请：通常需要进行许多低温尝试以获得高质量的无定形 冰。在细胞生物学应用中，该问题加剧了：细胞的热热扩散率低 对玻璃化过程中的冷却速率提出了严格的要求，从而限制了厚度 将样品的量表（<〜10μm）的 稀疏的种子细胞。冷冻验证过程将继续限制范围和吞吐量 冷冻 - 直到我们严格理解样品 - 晶体相互作用的流体动力学 在冷冻策划期间。一旦理解了此过程，我们就可以设计它以快速实现和 较厚样品的可再现冷却。优化冷冻验证过程将解决 几个关键的技术障碍，包括：（i）通过 提高样品制备的可重复性，（ii）扩大了通过 增加有资格进行冷冻策划的样品的厚度，甚至（iii）达到时间 - 通过精确时间间隔冷却样品对生物过程的纳米级成像解决 刺激后。 PI组成了一个合作团队，该团队唯一可以解决这些问题 通过使用计算和实验方法的组合来进行技术障碍 了解低温流并扩展（1）发展的冷冻策划功能 计算工具以模拟冷冻策划，（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 温度监测系统，并使用它使用生物学测试理论预测 样品。完成后，我们将进行以理论驱动的实验来评估 生物样品的最有希望的冷冻规程方案。新协议将增加 繁殖冷冻策划并将此技术扩展到较厚的样品，即 希望投资生物学相关的细胞组件和细胞 - 细胞通信。