School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
The Hamburg Centre for Ultrafast Imaging, Universität Hamburg, Hamburg, Germany.
Acta Crystallogr D Struct Biol. 2020 Apr 1;76(Pt 4):340-349. doi: 10.1107/S2059798320002958. Epub 2020 Mar 25.
Despite the great strides made in the field of single-particle cryogenic electron microscopy (cryo-EM) in microscope design, direct electron detectors and new processing suites, the area of sample preparation is still far from ideal. Traditionally, sample preparation involves blotting, which has been used to achieve high resolution, particularly for well behaved samples such as apoferritin. However, this approach is flawed since the blotting process can have adverse effects on some proteins and protein complexes, and the long blot time increases exposure to the damaging air-water interface. To overcome these problems, new blotless approaches have been designed for the direct deposition of the sample on the grid. Here, different methods of producing droplets for sample deposition are compared. Using gas dynamic virtual nozzles, small and high-velocity droplets were deposited on cryo-EM grids, which spread sufficiently for high-resolution cryo-EM imaging. For those wishing to pursue a similar approach, an overview is given of the current use of spray technology for cryo-EM grid preparation and areas for enhancement are pointed out. It is further shown how the broad aspects of sprayer design and operation conditions can be utilized to improve grid quality reproducibly.
尽管在显微镜设计、直接电子探测器和新的处理套件方面，单颗粒低温电子显微镜（cryo-EM）领域取得了巨大进展，但样品制备领域仍远非理想。传统上，样品制备涉及到印迹，这对于高分辨率，特别是对于 apoferritin 等行为良好的样品非常有效。然而，这种方法存在缺陷，因为印迹过程可能对某些蛋白质和蛋白质复合物产生不利影响，而且长时间的印迹会增加暴露于破坏性的气液界面的时间。为了克服这些问题，已经设计了新的无印迹方法，用于直接将样品沉积在网格上。在这里，比较了用于样品沉积的产生液滴的不同方法。使用气体动力学虚拟喷嘴，将小而高速的液滴沉积在 cryo-EM 网格上，这些液滴充分扩展，可用于高分辨率 cryo-EM 成像。对于那些希望采用类似方法的人，本文概述了喷雾技术在 cryo-EM 网格制备中的当前用途，并指出了需要改进的领域。进一步表明如何利用喷雾器设计和操作条件的广泛方面来可重复性地提高网格质量。
