Fast and accurate defocus 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, we propose a method for fast and accurate defocus (FADE) modulation during movie acquisition. 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 modulation by the typically undesired chromatic aberration of the objective lens. The simplicity, electrostatic principle and low electrical impedance of the device will allow fast switching speeds that will enable per-frame defocus values in cryo-EM movies. Researchers will be able to define custom defocus "recipes" and tailor the experiment for optimal information extraction from their 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.

Phase plates in the transmission electron microscope: operating principles and applications

“…Each of these could provide 19 Directions currently pursued to improve the resolution in (protein) structure determination by cryo electron microscopy include improved spherical aberration correctors (B-CORR) [206], high brightness cold field emission electron sources or a Schottky electron source with a monochromator and fast, high quantum efficiency detectors [207,257,281,282]. Focus modulation can improve the image interpretability at high spatial frequencies [198,208]. 20 The detrimental effect of spurious device charging can perhaps be reduced by holding the device at an elevated temperature.…”

“…13b and the possibility that a contamination of a similar nature may be present at the surface of the samples even at the < 5 × 10 −8 torr residual pressure in the Hitachi HF-3300 TEM. A biased film placed in the column also acts as an additional electrostatic lens, similar to [16,198].…”

“…Alternative, non-centro-symmetric differential phase-contrast methods were also tested in the context of biological sample imaging [52]. An achievement of great importance was the demonstration that, despite the contrast ringing artifacts, a thin-film 17 To obtain resolution directly, methods that improve the transfer of high spatial frequencies, such as direct electron detectors, high order aberration correction [206,207], effective focal length modulation [198] or high voltage modulation [208] could prove more fruitful [209]. Indeed, current knowledge indicates that such methods can be combined with HFPP.…”

“…More recently, the focus in high resolution cryo-TEM imaging and structure investigation has shifted toward increasing the efficiency of high spatial frequencies information transfer. One avenue for improving such transfer utilizes an additional einzel lens as proposed by [198,256], based on the concept reported in [208]. It is possible that adding a time-variable bias to HFPP could have the same effect as, for example [198,256], while not blocking the beam with hardware that is not electron transparent.…”

“…One avenue for improving such transfer utilizes an additional einzel lens as proposed by [198,256], based on the concept reported in [208]. It is possible that adding a time-variable bias to HFPP could have the same effect as, for example [198,256], while not blocking the beam with hardware that is not electron transparent. Simultaneous transfer of low spatial frequencies and high spatial frequencies should make it easier to align sub-images containing, for example, individual protein particles.…”