Extended Data Fig. 7: Improving the image acquisition speed over the time of the project. | Nature

Extended Data Fig. 7: Improving the image acquisition speed over the time of the project.

From: Atomic-resolution protein structure determination by cryo-EM

Extended Data Fig. 7

a, Several changes were made in the data acquisition scheme of the Titan Krios Mono/BCOR microscope. We started with manual data collection and then implemented several different EPU versions to increase the speed. EPU#1 was similar in speed to manual acquisition with one image per hole. EPU#2 required the automation of the C3 aperture and allowed us to take three images per hole. Only with EPU#3 we had a version allowing for automated re-centering of the monochromator which reduced the number of discarded images due to large dose variations. The currently highest speed in data acquisition is EPU#4 for which we reach roughly 100 movies per hour using the recording scheme shown in b) (only available since Dec 2019). Most data of this project was recorded with the EPU#3 scheme. b, To reach an acquisition speed of 100 micrographs/hour we move the stage only once to record data from 9 ice holes on a custom made EM grid (Quantifoil R1.2/1.0). Image/beam shift is applied to reach all 45 acquisition areas. The BCOR is tuned to compensate for image shift induced coma. Using this scheme we would also benefit significantly from a faster camera like the Falcon 4 (Thermo Fischer Scientific). Just by replacing the camera this would allow us to reach an almost 5 times higher recording speed of 500 micrographs per hour (F4 in a).

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