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中文摘要
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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。这个 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吞吐量 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。
英文摘要
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.
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HPF-X: High-pressure freezing with buffer exchange
  • 批准号:
    10704139
  • 项目类别:
  • 资助金额:
    $31.89万
  • 财政年份:
    2022
  • 负责人:
    Maxim Prigozhin
  • 依托单位:
Engineering fluid dynamics of cryo-plunging for improved vitrification
  • 批准号:
    10430822
  • 项目类别:
  • 资助金额:
    $22.55万
  • 财政年份:
    2022
  • 负责人:
    Maxim Prigozhin
  • 依托单位:
海外基金