Mendez Joshua H, Mehrani Atousa, Randolph Peter, Stagg Scott
Department of Physics, Florida State University, 77 Chieftan Way, Tallahassee, FL 32306, USA.
Department of Chemistry and Biochemistry, Florida State University, 95 Chieftain Way, Tallahassee, FL 32306, USA.
IUCrJ. 2019 Oct 24;6(Pt 6):1007-1013. doi: 10.1107/S2052252519012661. eCollection 2019 Nov 1.
Direct electron detectors (DEDs) have revolutionized cryo-electron microscopy (cryo-EM) by facilitating the correction of beam-induced motion and radiation damage, and also by providing high-resolution image capture. A new-generation DED, the DE64, has been developed by Direct Electron that has good performance in both integrating and counting modes. The camera has been characterized in both modes in terms of image quality, throughput and resolution of cryo-EM reconstructions. The modulation transfer function, noise power spectrum and detective quantum efficiency (DQE) were determined for both modes, as well as the number of images per unit time. Although the DQE for counting mode was superior to that for integrating mode, the data-collection throughput for this mode was more than ten times slower. Since throughput and resolution are related in single-particle cryo-EM, data for apoferritin were collected and reconstructed using integrating mode, integrating mode in conjunction with a Volta phase plate (VPP) and counting mode. Only the counting-mode data resulted in a better than 3 Å resolution reconstruction with similar numbers of particles, and this increased performance could not be compensated for by the increased throughput of integrating mode or by the increased low-frequency contrast of integrating mode with the VPP. These data show that the superior image quality provided by counting mode is more important for high-resolution cryo-EM reconstructions than the superior throughput of integrating mode.
直接电子探测器（DEDs）通过促进束流诱导运动和辐射损伤的校正，以及提供高分辨率图像捕获，彻底改变了冷冻电子显微镜（cryo-EM）。Direct Electron公司开发了新一代DED——DE64，它在积分模式和计数模式下均具有良好的性能。该相机在这两种模式下均从冷冻电镜重建的图像质量、通量和分辨率方面进行了表征。确定了两种模式下的调制传递函数、噪声功率谱和探测量子效率（DQE），以及单位时间内的图像数量。尽管计数模式下的DQE优于积分模式，但该模式下的数据采集通量慢了十多倍。由于在单颗粒冷冻电镜中通量和分辨率相关，因此使用积分模式、结合Volta相板（VPP）的积分模式和计数模式收集并重建了脱铁铁蛋白的数据。只有计数模式的数据在粒子数量相近的情况下，重建分辨率优于3 Å，而积分模式通量的增加或结合VPP的积分模式低频对比度的增加都无法弥补这种性能提升。这些数据表明，对于高分辨率冷冻电镜重建而言，计数模式提供的卓越图像质量比积分模式的卓越通量更为重要。
