Danev Radostin, Yanagisawa Haruaki, Kikkawa Masahide
Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan.
Microscopy (Oxf). 2021 Nov 24;70(6):487-497. doi: 10.1093/jmicro/dfab016.
The increasing popularity and adoption rate of cryo-electron microscopy (cryo-EM) is evidenced by a growing number of new microscope installations around the world. The quality and reliability of the instruments improved dramatically in recent years, but site-specific issues or unnoticed problems during installation could undermine productivity. Newcomers to the field may also have limited experience and/or low confidence in the capabilities of the equipment or their own skills. Therefore, it is recommended to perform an initial test of the complete cryo-EM workflow with an 'easy' test sample, such as apoferritin, before starting work with real and challenging samples. Analogous test experiments are also recommended for the quantification of new data acquisition approaches or imaging hardware. Here, we present the results from our initial tests of a recently installed Krios G4 electron microscope equipped with two latest generation direct electron detector cameras-Gatan K3 and Falcon 4. Three beam-image shift-based data acquisition strategies were also tested. We detail the methodology and discuss the critical parameters and steps for performance testing. The two cameras performed equally, and the single- and multi-shot per-hole acquisition schemes produced comparable results. We also evaluated the effects of environmental factors and optical flaws on data quality. Our results reaffirmed the exceptional performance of the software aberration correction in Relion in dealing with severe coma aberration. We hope that this work will help cryo-EM teams in their testing and troubleshooting of hardware and data collection approaches.
全球范围内新安装的冷冻电子显微镜（cryo-EM）数量不断增加，这证明了其越来越受欢迎以及采用率不断提高。近年来，仪器的质量和可靠性有了显著提升，但安装过程中特定地点的问题或未被注意到的问题可能会影响工作效率。该领域的新手在设备能力或自身技能方面的经验可能也有限，信心也较低。因此，建议在开始处理真实且具有挑战性的样品之前，先用“简单”的测试样品（如脱铁铁蛋白）对完整的冷冻电子显微镜工作流程进行初步测试。对于新的数据采集方法或成像硬件的量化，也建议进行类似的测试实验。在此，我们展示了对最近安装的配备两台最新一代直接电子探测器相机（Gatan K3和Falcon 4）的Krios G4电子显微镜进行初步测试的结果。还测试了三种基于束流图像偏移的数据采集策略。我们详细介绍了方法，并讨论了性能测试的关键参数和步骤。两台相机表现相当，每孔单帧和多帧采集方案产生的结果相当。我们还评估了环境因素和光学缺陷对数据质量的影响。我们的结果再次证实了Relion软件像差校正处理严重彗形像差时的卓越性能。我们希望这项工作将有助于冷冻电子显微镜团队对硬件和数据采集方法进行测试及故障排除。
