A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic “silver bullet” for improving the quality of every cryoEM sample preparation.

许多新技术正在开发中，以提高冷冻电子显微镜 (cryoEM) 网格制备的质量和再现性。在这里，我们系统地研究了使用三种不同的玻璃化设备（Vitrobot、chameleon 和时间分辨冷冻电镜设备）在不同时间尺度上制备三种大分子复合物的过程，包括在 6 毫秒内制作网格（迄今为止最快的报告），以询问粒子不同时间点空气-水界面的行为。结果表明，不同的大分子复合物可以以高度可变的方式响应冷冻电镜样品制备过程中形成的薄膜环境，这揭示了为什么冷冻电镜样品制备难以优化。我们证明，缩短样品应用和玻璃化之间的时间只是提高冷冻电镜网格质量的一种工具，但它不太可能成为提高每个冷冻电镜样品制备质量的通用“灵丹妙药”。