High-quality, high-throughput cryo-electron microscopy data collection via beam tilt and astigmatism-free beam-image shift

Assessing the JEOL CRYO ARM 300 for high-throughput automated single-particle cryo-EM in a multiuser environment

“…Faster detectors will reduce image acquisition time. Our preliminary estimates show that on a K3 detector similar collection settings can reduce the image recording time from 20 to around 6 s. Data collection throughput can be further significantly increased by introducing aberration-corrected beam-image shift conditions to record images from multiple holes without moving the stage (Wu et al, 2019). Ice contamination rates.…”

“…However, for the more commonly encountered conditions for which the ice thickness exceeds 50 nm the effect of the ice contamination is not significant. Moreover, with the increasing rate of data collection due to faster detectors and the use of coma-corrected imaging (Wu et al, 2019), the duration of the data collection rarely needs to be longer than 48 h, making the negative impact of ice contamination less important.…”

“…This is particularly true for difficult projects, where regular high-resolution data collection is required. The efforts to accelerate and facilitate structural projects include establishing a constantly growing number of cryo-EM facilities with supervised access to automated data collection, increasing throughput of data collection Wu et al, 2019), designing cheaper and better screening microscopes (Naydenova et al, 2019), and improving sample preparation methodologies (Ravelli et al, 2019;Razinkov et al, 2016).…”

High-resolution cryo-EM reconstructions in the presence of substantial aberrations

“…The success of this approach and similar approaches requires co-alignment of the optical axes for multiple lenses, and if the alignment procedures are not properly executed, coma and trefoil may be present and affect the quality of the SPR results, and yet only manifest in specialized analyses (Uhlemann & Haider, 1998;Mastronarde, 2005;Suloway et al, 2005;Cheng et al, 2018;Zivanov et al, 2020). Furthermore, these and additional, higher order antisymmetric aberrations are induced when data are acquired using the beam-image shift method (Mastronarde, 2005;Suloway et al, 2005;Cheng et al, 2018;Wu et al, 2019), in which coordinated electronic shifts of an illuminating beam and an image are used to navigate away from the optical axis. We restrict our discussion here to axial aberrations, but with the understanding that these aberrations will have different values in different positions of an optical system for data acquired with the beam-image shift method, except for coma, which can be compensated for by the beam tilt (Wu et al, 2019;Glaeser et al, 2011).…”

“…Furthermore, these and additional, higher order antisymmetric aberrations are induced when data are acquired using the beam-image shift method (Mastronarde, 2005;Suloway et al, 2005;Cheng et al, 2018;Wu et al, 2019), in which coordinated electronic shifts of an illuminating beam and an image are used to navigate away from the optical axis. We restrict our discussion here to axial aberrations, but with the understanding that these aberrations will have different values in different positions of an optical system for data acquired with the beam-image shift method, except for coma, which can be compensated for by the beam tilt (Wu et al, 2019;Glaeser et al, 2011).…”