Hypothesis for a mechanism of beam-induced motion in cryo-electron microscopy

Abstract

Estimates of heat-transfer rates during plunge-cooling and the patterns of ice observed in cryo-EM samples indicate that the grid bars cool much more slowly than do the support foil and sample near the middle of the grid openings. The resulting transient temperature differences generate transient tensile stresses in the support foil. Most of this foil stress develops while the sample is liquid and cooling toward its glass transition T g, and so does not generate tensile sample stress. As the grid bars continue cooling towards the cryogen temperature and contracting, the tensile stress in the foil is released, placing the sample in compressive stress. Radiation-induced creep in the presence of this compressive stress should generate a doming of the sample in the foil openings, as is observed experimentally. Crude estimates of the magnitude of the doming that may be generated by this mechanism are consistent with observation. Several approaches to reducing beam-induced motion are discussed.

High-resolution single-particle cryo-EM of samples vitrified in boiling nitrogen

“…8 stressed foil) and when the grid and foil have both cooled to the temperature of the liquid cryogen. The overall magnitude of the observed motion is consistent with rough estimates of grid-foil temperature differences during cooling (Thorne, 2020). Doming motion is reduced if the liquid ethane temperature used for sample vitrification is increased [e.g.…”

“…At low total doses where motion is most rapid with dose, the RMS magnitude of particle motion within a given foil hole is (to within experimental uncertainties) proportional to the diameter of the hole (Naydenova et al, 2020; see Fig. S8), as predicted by Thorne (2020): the doming amplitude h / a , where a is the hole diameter and is the (dimensionless) foil strain released between when the sample initially vitrifies (on the tensile-research papers IUCrJ (2021). 8 stressed foil) and when the grid and foil have both cooled to the temperature of the liquid cryogen.…”

Devitrification reduces beam-induced movement in cryo-EM

“…Beam-induced movement is most likely caused by pre-existing mechanical stress frozen into the sample that is released upon electron irradiation (Glaeser, 2016;Vinothkumar & Henderson, 2016;Thorne, 2020;Naydenova et al, 2020). By comparison, specimen charging (Glaeser, 2016) appears to play only a minor role (Russo & Henderson, 2018a,b).…”

Current limitations to high-resolution structure determination by single-particle cryoEM

“…4 b ) (Wieferig et al ., 2021). A theoretical analysis of beam-induced movement based on heat transfer rates during cooling (Thorne, 2020) makes a number of suggestions of how the movement can be reduced, e.g. by appropriate choice of materials for the grid and support film.…”