Abstract
Current data collection strategies in electron cryo-microscopy (cryo-EM) record multiframe movies with static optical settings. This limits the number of adjustable parameters that can be used to optimize the experiment. Here, a method for fast and accurate defocus (FADE) modulation during movie acquisition is proposed. It uses the objective lens aperture as an electrostatic pole that locally modifies the electron beam potential. The beam potential variation is converted to defocus change by the typically undesired chromatic aberration of the objective lens. The simplicity, electrostatic principle and low electrical impedance of the device allow fast switching speeds that will enable per-frame defocus modulation of cryo-EM movies. Researchers will be able to define custom defocus 'recipes' and tailor the experiment for optimal information extraction from the sample. The FADE method could help to convert the microscope into a more dynamic and flexible optical platform that delivers better performance in cryo-EM single-particle analysis and electron cryo-tomography.
Highlights
The introduction of direct electron detectors (DEDs) revolutionized the cryo-EM field
It is connected to the fast and accurate defocus (FADE) control unit, which generates a synchronized defocus modulation pattern and applies it during exposure
The controller outputs the pattern as a digital stream to a high-precision digital-to-analog converter (DAC)
Summary
The introduction of direct electron detectors (DEDs) revolutionized the cryo-EM field. With their improved signal-tonoise ratio and multiframe movie capabilities, these new cameras have led to the ‘resolution revolution’ that propelled cryo-EM from a niche technique to a powerful mainstream structural method (Rivera-Calzada & Carroni, 2019). The movie acquisition capability of DEDs enables alignment and registration of individual movie frames to counteract one of the main resolution-limiting factors in cryo-EM, beam-induced specimen motion. When irradiated, frozenhydrated cryo-specimens experience changes in their shape that cause image blurring (Brilot et al, 2012), presumably due to release of mechanical stresses built up during the freezing process (Thorne, 2019). Alignment and averaging of the movie frames, called motion correction, can minimize the blurring
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