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.


Captions for supplementary movies
Supplementary Movies are available at: https://www.dropbox.com/sh/sb8wval7cxt7v1p/AACONjlQgO8s_lLtJmKPYuEYa?dl=0 Supplementary Movie 1: Effect of specimen charging on Quantifoil in overfocus condition shown by DIFF images. The beam was parallel on vitreous specimen with the beam diameter (800 nm) smaller than the hole diameter (1.2 um). The electron beam flux on the sample was at 0.38 e -/Å 2 /s. The beam was blocked with the pre-specimen beam shutter before exposing the sample for recording showing as the black frames at the beginning of the movie.
Supplementary Movie 2: Effect of specimen charging on UltrAuFoil in overfocus condition shown by DIFF images. The beam was parallel on vitreous specimen with the beam diameter (800 nm) smaller than the hole diameter (1.2 um). The electron beam flux on the sample was at 0.38 e -/Å 2 /s. The beam was blocked with the pre-specimen beam shutter before exposing the sample for recording showing as the black frames at the beginning of the movie.
Supplementary Movie 3: Effect of specimen charging on Quantifoil in underfocus condition shown by DIFF images. The beam was parallel on vitreous specimen with the beam diameter (800 nm) smaller than the hole diameter (1.2 um). The electron beam flux on the sample was at 0.38 e -/Å 2 /s. The beam was blocked with the pre-specimen beam shutter before exposing the sample for recording showing as the black frames at the beginning of the movie.
Supplementary Movie 4: Effect of specimen charging on UltrAuFoil in underfocus condition shown by DIFF images. The beam was parallel on vitreous specimen with the beam diameter (800 nm) smaller than the hole diameter (1.2 um). The electron beam flux on the sample was at 0.38 e -/Å 2 /s. The beam was blocked with the pre-specimen beam shutter before exposing the sample for recording showing as the black frames at the beginning of the movie.
Supplementary Movie 5: Effect of specimen charging on cross-grating in overfocus condition shown by DIFF images. The beam was parallel with the beam diameter of 800 nm. The electron beam flux on the sample was at 0.38 e -/Å 2 /s. The beam was blocked with the pre-specimen beam shutter before exposing the sample for recording showing as the black frames at the beginning of the movie.
Supplementary Movie 6: Effect of specimen charging on cross-grating in underfocus condition shown by DIFF images. The beam was parallel with the beam diameter of 800 nm. The electron beam flux on the sample was at 0.38 e -/Å 2 /s. The beam was blocked with the pre-specimen beam shutter before exposing the sample for recording showing as the black frames at the beginning of the movie. Supplementary Movie 7: Effect of specimen charging on Quantifoil in overfocus condition upon stage move shown by DIFF images. The beam was parallel on vitreous specimen with the beam diameter (800 nm) smaller than the hole diameter (1.2 um). The electron beam flux on the sample was at 0.38 e -/Å 2 /s. The beam was blocked with the pre-specimen beam shutter before exposing the sample for recording showing as the black frames at the beginning of the movie. The beam was completely within the foil hole on ice at the beginning of irradiation. After 5 s, the beam partially moved to one side of the carbon foil, followed by movement onto the carbon foil for 5 s.
The beam then moved back to the edge of the foil for 5 s, then to the foil hole for another 5 s. The beam quickly scanned through the foil and remained at the edge of the carbon foil.
Supplementary Movie 8: Effect of specimen charging on Quantifoil in underfocus condition upon stage move shown by DIFF images. The beam was parallel on vitreous specimen with the beam diameter (800 nm) smaller than the hole diameter (1.2 um). The electron beam flux on the sample was at 0.38 e -/Å 2 /s. The beam was blocked with the pre-specimen beam shutter before exposing the sample for recording showing as the black frames at the beginning of the movie. The beam was completely within the foil hole on ice at the beginning of irradiation. After 5 s, the beam partially moved to one side of the carbon foil, followed by movement onto the carbon foil for 5 s.
The beam then moved back to the edge of the foil for 5 s, then to the foil hole for another 5 s. The beam quickly scanned through the foil and remained at the edge of the carbon foil.
Supplementary Movie 9: Effect of specimen charging on UltrAuFoil in overfocus condition upon stage move shown by DIFF images. The beam was parallel on vitreous specimen with the beam diameter (800 nm) smaller than the hole diameter (1.2 um). The electron beam flux on the sample was at 0.38 e -/Å 2 /s. The beam was blocked with the pre-specimen beam shutter before exposing the sample for recording showing as the black frames at the beginning of the movie. The beam was completely within the foil hole on ice at the beginning of irradiation. After 5 s, the beam partially moved to one side of the gold foil, followed by movement onto the gold foil for 5 s. The beam then moved back to the edge of the foil for 5 s, then to the foil hole for another 5 s. The beam quickly scanned through the foil and remained at the edge of the gold foil.
Supplementary Movie 10: Effect of specimen charging on UltrAuFoil in underfocus condition upon stage move shown by DIFF images. The beam was parallel on vitreous specimen with the beam diameter (800 nm) smaller than the hole diameter (1.2 um). The electron beam flux on the sample was at 0.38 e -/Å 2 /s. The beam was blocked with the pre-specimen beam shutter before exposing the sample for recording showing as the black frames at the beginning of the movie. The beam was completely within the foil hole on ice at the beginning of irradiation. After 5 s, the beam partially moved to one side of the gold foil, followed by movement onto the gold foil for 5 s. The beam then moved back to the edge of the foil for 5 s, then to the foil hole for another 5 s. The beam quickly scanned through the foil and remained at the edge of the gold foil. The stage movement simulates the beam transitioning from the sample to the carbon foil.
Supplementary Movie 17: Effect of specimen charging on graphene-coated UltrAuFoil in overfocus condition upon stage move shown by DIFF images. The beam was parallel on vitreous specimen with the and a beam diameter (800 nm) smaller than the hole diameter (1.2 um). The electron beam flux on the sample was at 0.38 e -/Å 2 /s. The beam was blocked with the pre-specimen beam shutter before exposing the sample for recording showing as the black frames at the beginning of the movie. The beam was completely within the foil hole on ice at the beginning of irradiation. The stage movement simulates the beam transitioning from the sample to the gold foil.
Supplementary Movie 18: Effect of specimen charging on graphene-coated UltrAuFoil in underfocus condition upon stage move shown by DIFF images. The beam was parallel on vitreous specimen with the and a beam diameter (800 nm) smaller than the hole diameter (1.2 um). The electron beam flux on the sample was at 0.38 e -/Å 2 /s. The beam was blocked with the pre-specimen beam shutter before exposing the sample for recording showing as the black frames at the beginning of the movie. The beam was completely within the foil hole on ice at the beginning of irradiation. The stage movement simulates the beam transitioning from the sample to the gold foil.
Supplementary Movie 19: Effect of specimen charging on Quantifoil in imaging mode. A micrograph of the BfrB sample with a concentration of 50 mg/ml on 1.2/1.3 Quantifoil grids was obtained at a magnification of 78,000× using a Falcon III at 200 kV. The data were collected with the beam not hitting the carbon foil, with a flux of 30 e-/Å2/s for 1 s using a 20 um aperture (beam diameter of 800 nm), and then repeated at the same position using a 50 um aperture (beam diameter of 1.9 um). A movie was generated by performing a running average (1-3, 2-4, …) of the fractions (40 fractions/s) and applying a Gaussian function (sigma = 3). No objective aperture was used.