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.

项目总结摘要 该项目的长期目标是改进冷冻玻璃化样品的制备方法 低温电子显微镜(Cryo-EM)和体层摄影术(Cryo-ET)的重复性 以及样品厚度限制。低温电子显微镜是一种很有前途的亚细胞观察方法 具有分子分辨率的原位组装。然而，冷冻-EM受到 冷冻玻璃化过程施加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放入低温液体中来实现的。这 即使在结构生物学中，低温坠落的过程也是出了名的不可重现 应用：要获得高质量的无定形晶体，通常需要多次低温下沉尝试 冰。在细胞生物学应用中，问题变得更加严重：细胞的低热扩散率 在玻璃化过程中对冷却速度提出了严格的要求，限制了厚度 样品的微米级(~10μm)，这限制了这项技术在 种子稀少的细胞。冷冻玻璃化过程将继续限制范围和产量 直到我们严格理解样品-制冷剂相互作用的流体动力学 在低温俯冲过程中。一旦了解了这一过程，我们就可以对其进行设计，以实现快速和 可重复冷却较厚的样品。优化冷冻玻璃化过程将解决 若干关键技术障碍，包括：(1)通过以下方式实现高通量样品处理 增加样品制备的重现性；(2)扩大冷冻范围： 增加符合低温下沉条件的样品的厚度，甚至(Iii)实现时间- 通过以精确的时间间隔冷却样品来解析生物过程的纳米级成像 在刺激之后。PI组成了一个协作团队，该团队的独特定位是解决这些问题 使用计算方法和实验方法相结合的技术壁垒 了解低温流动，通过(1)发展，扩大低温下沉能力 用于模拟低温俯冲的计算工具，(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)用生物学验证理论预测 样本。完成后，我们将进行理论驱动的实验，评估 最有希望的生物样品低温降温方案。新的协议将增加 低温下沉的重现性，并将这项技术扩展到更厚的样品，这是 适用于生物相关细胞组件和细胞间通讯的研究。