Research Institute of Agriculture and Life Sciences, Center for Food and Bioconvergence, Department of Agricultural Biotechnology, Interdisciplinary Programs in Agricultural Genomics, College of Agriculture and Life Sciences (CALS), Seoul National Un.
These authors equally contributed to this work.
Mol Cells. 2023 Sep 30;46(9):538-544. doi: 10.14348/molcells.2023.0058. Epub 2023 Aug 2.
The formation of uniform vitreous ice is a crucial step in the preparation of samples for cryogenic electron microscopy (cryo-EM). Despite the rapid technological progress in EM, controlling the thickness of vitreous ice on sample grids with reproducibility remains a major obstacle to obtaining high-quality data in cryo-EM imaging. The commonly employed classical blotting process faces the problem of excess water that cannot be absorbed by the filter paper, resulting in the formation of thick and heterogeneous ice. In this study, we propose a novel approach that combines the recently developed nanowire self-wicking technique with the classical blotting method to effectively control the thickness and homogeneity of vitrified ice. With simple procedures, we generated a copper oxide spike (COS) grid by inducing COSs on commercially available copper grids, which can effectively remove excess water during the blotting procedure without damaging the holey carbon membrane. The ice thickness could be controlled with good reproducibility compared to non-oxidized grids. Incorporated into other EM techniques, our new modification method is an effective option for obtaining high-quality data during cryo-EM imaging.
形成均匀的玻璃态冰是低温电子显微镜（cryo-EM）制备样品的关键步骤。尽管在 EM 方面取得了快速的技术进步，但在具有重现性的情况下控制样品网格上玻璃态冰的厚度仍然是获得 cryo-EM 成像高质量数据的主要障碍。常用的经典印迹过程面临滤纸无法吸收的多余水的问题，导致形成厚而不均匀的冰。在这项研究中，我们提出了一种新方法，将最近开发的纳米线自吸技术与经典印迹方法相结合，有效地控制玻璃态冰的厚度和均匀性。通过简单的程序，我们在市售的铜网格上诱导出氧化铜尖峰（COS），从而生成了氧化铜尖峰（COS）网格，该网格可以在印迹过程中有效去除多余的水，而不会损坏有孔碳膜。与未氧化的网格相比，冰的厚度可以具有良好的重现性来进行控制。与其他 EM 技术结合，我们的新改进方法是在 cryo-EM 成像中获得高质量数据的有效选择。
