Department of Mechanical and Aerospace Engineering, Monash University, Clayton, Victoria, Australia.
Ramaciotti Centre for Electron Microscopy, Monash University, Clayton, Victoria, Australia.
J Struct Biol. 2018 Aug;203(2):94-101. doi: 10.1016/j.jsb.2018.03.012. Epub 2018 Apr 6.
Cryo-Electron Microscopy (cryo-EM) has become an invaluable tool for structural biology. Over the past decade, the advent of direct electron detectors and automated data acquisition has established cryo-EM as a central method in structural biology. However, challenges remain in the reliable and efficient preparation of samples in a manner which is compatible with high time resolution. The delivery of sample onto the grid is recognized as a critical step in the workflow as it is a source of variability and loss of material due to the blotting which is usually required. Here, we present a method for sample delivery and plunge freezing based on the use of Surface Acoustic Waves to deploy 6-8 µm droplets to the EM grid. This method minimises the sample dead volume and ensures vitrification within 52.6 ms from the moment the sample leaves the microfluidics chip. We demonstrate a working protocol to minimize the atomised volume and apply it to plunge freeze three different samples and provide proof that no damage occurs due to the interaction between the sample and the acoustic waves.
冷冻电子显微镜（cryo-EM）已成为结构生物学中不可或缺的工具。在过去的十年中，直接电子探测器和自动化数据采集的出现使得 cryo-EM 成为结构生物学的核心方法。然而，在以兼容高时间分辨率的方式可靠且高效地制备样品方面仍然存在挑战。将样品递送到网格上被认为是工作流程中的一个关键步骤，因为由于通常需要的吸取，它是产生变异性和材料损失的来源。在这里，我们提出了一种基于使用表面声波将 6-8µm 液滴部署到 EM 网格的样品传递和骤冷方法。该方法最小化了样品死体积，并确保在样品离开微流控芯片后的 52.6ms 内实现玻璃化。我们演示了一个工作协议来最小化雾化体积，并将其应用于三种不同的骤冷样品，并证明由于样品与声波之间的相互作用不会造成损坏。
