3D simulation of sample vitrification in cryo-plunging*

Biological sample preparation is vital for cryo-electron microscopy (cryo-EM) to achieve molecular resolution in structural biological applications. However, the vitrification process of plunging the sample into a cryogenic fluid suffers from poor reproducibility and limitations in sample thickness. In this collaborative project, we address these technical barriers with experiment-based simulations and simulation-driven experiments. We have developed a 3D computational tool to simulate the fluid dynamics and temperature profile during cryo-plunging. This tool is integrated with a block-structured adaptive mesh refinement software framework (AMReX) and highly parallelized for efficient simulations. Our simulations accurately replicate the sample–cryogen interaction and have an excellent match with experimental thermocouple data at different plunging speeds. In addition, we now have visualizations of the spatial temperature profile of each biological sample on the EM grid, which are hard to capture experimentally. Our tool empowers systematic exploration of the design space to optimize cryo-plunging protocols for faster cooling of thicker samples and enable a time-resolved temperature monitoring system.