Charging of Vitreous Samples in Cryogenic Electron Microscopy Mitigated by Graphene

Cryogenic electron
microscopy can provide high-resolution 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 graphene-coated 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.