Engineering fluid dynamics of cryo-plunging for improved vitrification

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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.