Charging of Vitreous Samples in Cryogenic Electron Microscopy Mitigated by Graphene

Yue Zhang*, J. Paul van Schayck, Adriain Pedrazo-Tardajos, Nathalie Claes, Willem E. M. Noteborn, Peng-Han Lu, Hans Duimel, Rafal E. Dunin-Borkowski*, Sara Bals, Peter J. Peters, Raimond B. G. Ravelli

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review


Cryogenic electronmicroscopy can provide high-resolution reconstructionsof macromolecules embedded in a thin layer of ice from which atomicmodels can be built de novo. However, the interactionbetween the ionizing electron beam and the sample results in beam-inducedmotion and image distortion, which limit the attainable resolutions.Sample charging is one contributing factor of beam-induced motionsand image distortions, which is normally alleviated by including partof the supporting conducting film within the beam-exposed region.However, routine data collection schemes avoid strategies wherebythe beam is not in contact with the supporting film, whose rationaleis not fully understood. Here we characterize electrostatic chargingof vitreous samples, both in imaging and in diffraction mode. We mitigatesample charging by depositing a single layer of conductive grapheneon top of regular EM grids. We obtained high-resolution single-particleanalysis (SPA) reconstructions at 2 & ANGS; when the electron beamonly irradiates the middle of the hole on graphene-coated grids, usingdata collection schemes that previously failed to produce sub 3 & ANGS;reconstructions without the graphene layer. We also observe that theSPA data obtained with the graphene-coated grids exhibit a higher b factor and reduced particle movement compared to dataobtained without the graphene layer. This mitigation of charging couldhave broad implications for various EM techniques, including SPA andcryotomography, and for the study of radiation damage and the developmentof future sample carriers. Furthermore, it may facilitate the explorationof more dose-efficient, scanning transmission EM based SPA techniques.
Original languageEnglish
Pages (from-to)15836-15846
Number of pages11
JournalACS Nano
Issue number16
Early online date1 Aug 2023
Publication statusPublished - 2 Aug 2023


  • charging
  • vitreous samples
  • single-particleanalysis
  • graphene
  • cryogenic transmission electronmicroscopy


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