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.

中文翻译：

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硬件和数据采集策略的 Cryo-EM 性能测试

低温电子显微镜 (cryo-EM) 的日益普及和采用率从世界各地越来越多的新显微镜装置得到证明。近年来，仪器的质量和可靠性有了显着提高，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降低生产力。该领域的新手也可能经验有限和/或对设备的能力或他们自己的技能缺乏信心。因此，建议在开始使用真实且具有挑战性的样品之前，使用“简单”的测试样品（例如脱铁铁蛋白）对完整的冷冻电镜工作流程进行初步测试。类似的测试实验也被推荐用于量化新的数据采集方法或成像硬件。这里，我们展示了我们对最近安装的 Krios G4 电子显微镜的初步测试结果，该显微镜配备了两个最新一代的直接电子探测器相机 - Gatan K3 和 Falcon 4。还测试了三种基于光束图像位移的数据采集策略。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。两台相机的性能相同，单孔和多孔采集方案产生了可比较的结果。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 软件像差校正在处理严重彗形像差方面的卓越性能。我们希望这项工作能够帮助 cryo-EM 团队对硬件和数据收集方法进行测试和故障排除。