Abstract
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.
Highlights
The technology used in recording images for cryo-electron microscopy has evolved from the use of photographic film to charge-coupled devices (CCD) and recently direct electron detector (DED) cameras
These are difficult quantities to measure experimentally, so detective quantum efficiency (DQE) can be reformulated in terms of the modulation transfer function (MTF) of the camera and its noise power spectrum (NPS) using the equation DQE(!) = DQE(0) Â MTF2(!)/ NPSnormalized(!) (McMullan et al, 2009)
The DE64 in counting mode operates at a frame rate of 141 frames per second and NPS were calculated with dose rates of 2, 2.86 and 3.39 eÀ per pixel per second
Summary
The technology used in recording images for cryo-electron microscopy (cryo-EM) has evolved from the use of photographic film to charge-coupled devices (CCD) and recently direct electron detector (DED) cameras. Direct detectors can operate in one of two modes: integrating mode, in which electron hits deposit some amount of charge that is built up over the course of an exposure, and counting mode, in which individual electron hits are ‘counted’ and the counts are summed over the exposure. Counting mode eliminates the Landau noise that results from individual electrons depositing different amounts of energy on the detector. On the other hand, counting requires a very weak beam and long exposure times, which can result in lower throughput. These detector advancements, together with the development of new software, have helped cryo-EM to break the 3 Aresolution boundary (Herzik et al, 2017). A new-generation DED called the DE64 built by Direct Electron promises good performance when used in either integrating or counting mode.
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