Charging of Vitreous Samples in Cryogenic Electron Microscopy Mitigated by Graphene

Abstract

Cryogenic electron microscopy can provide highresolution reconstructions of macromolecules embedded in a thin layer of ice from which atomic models can be built de novo. However, the interaction between the ionizing electron beam and the sample results in beam-induced motion and image distortion, which limit the attainable resolutions. Sample charging is one contributing factor of beam-induced motions and image distortions, which is normally alleviated by including part of the supporting conducting film within the beam-exposed region. However, routine data collection schemes avoid strategies whereby the beam is not in contact with the supporting film, whose rationale is not fully understood. Here we characterize electrostatic charging of vitreous samples, both in imaging and in diffraction mode. We mitigate sample charging by depositing a single layer of conductive graphene on top of regular EM grids. We obtained high-resolution single-particle analysis (SPA) reconstructions at 2 Å when the electron beam only irradiates the middle of the hole on graphene-coated grids, using data collection schemes that previously failed to produce sub 3 Å reconstructions without the graphene layer. We also observe that the SPA data obtained with the graphenecoated grids exhibit a higher b factor and reduced particle movement compared to data obtained without the graphene layer. This mitigation of charging could have broad implications for various EM techniques, including SPA and cryotomography, and for the study of radiation damage and the development of future sample carriers. Furthermore, it may facilitate the exploration of more dose-efficient, scanning transmission EM based SPA techniques.

Liquid phase electron microscopy of bacterial ultrastructure

“…The top graphene layer for encapsulation was fabricated using a polymer-free transfer method ( 55 ), which provides a flat top surface to the hydrated environment which then adapts to the morphology of the target. The main steps are described in detail in refs ( 56 ) and ( 25 ) to prepare the base TEM grid and the creation of the encapsulated environments respectively ( 57 ).…”

Pillar data-acquisition strategies for cryo-electron tomography of beam-sensitive biological samples

“…This is known as the 'jump-at-start' issue for the traditional bi-directional tilt scheme where, starting from zero tilt, the negative tilt branch is collected followed by the positive tilt branch (Hagen et al, 2017). Additionally, charging of the sample in the electron beam also results in beam-induced motion and image distortion (Zhang et al, 2023). One of the reasons that the classic dose-symmetric scheme is preferred for planar samples is that the fluence varies smoothly between adjacent images.…”