Fast and accurate defocus modulation for improved tunability of cryo-EM experiments

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.

The promise and the challenges of cryo‐electron tomography

“…An idea put forward by Danev et al . [165] is to use defocus modulations to optimally extract sample information. Inserting a small electrostatic lens at the back focal plane would allow control and fast tuning of the defocus parameter during acquisition, which can be used as an optical dose‐dependent filter that follows the gradual demise of high‐resolution structural features.…”

Cryogenic electron ptychographic single particle analysis with wide bandwidth information transfer

“…4a), which is attributed to the zero-crossings and contrast reversal in the TEM CTF. These could however, be compensated in the 3D maps by implementation of CTF correction 11,51 , use of a wide range of defocus values 52 and collection of data from a sufficiently large number of particles with varying defocus values. However, the FSC curve of the 3D maps using cryo-EPty SPA shows a gradual decrease with spatial frequency with no oscillations, due to the continuous positive nature of the ptychographic transfer function over its entire spatial frequency range 17 .…”