Temporal dynamics of charge buildup in cryo-electron microscopy

Abstract

It is well known that insulating samples can accumulate electric charges from exposure to an electron beam. How the accumulation of charge affects imaging parameters and sample stability in transmission electron microscopy is poorly understood. To quantify these effects, it is important to know how the charge is distributed within the sample and how it builds up over time. In the present study, we determine the spatial distribution and temporal dynamics of charge accumulation on vitreous ice samples with embedded proteins through a combination of modeling and Fresnel diffraction experiments. Our data reveal a rapid evolution of the charge state on ice upon initial exposure to the electron beam accompanied by charge gradients at the interfaces between ice and carbon films. We demonstrate that ice film movement and charge state variations occur upon electron beam exposure and are dose-rate dependent. Both affect the image defocus through a combination of sample height changes and lensing effects. Our results may be used as a guide to improve sample preparation, data collection, and data processing for imaging of dose-sensitive samples.

Keywords: Cryo-electron microscopy; Defocused diffraction; Fresnel diffraction; Ice; Specimen charging.

Charging affects image defocus and sample motion dynamically

Fig. 1

Selected frames from a movie of the central defocused diffraction spot from a 913 nm diameter and 1.51${\mathrm{e}}^{-}/{\mathrm{\AA }}^2/\mathrm{s}$ electron dose-rate beam centered on an ice film recorded at 40 fps. a) The first frame (0.04${\mathrm{e}}^{-}/{\mathrm{\AA }}^2$ accumulated dose), b) the second frame (0.08${\mathrm{e}}^{-}/{\mathrm{\AA }}^2$), c) the fifth frame (0.19${\mathrm{e}}^{-}/{\mathrm{\AA }}^2/\mathrm{s}$), and d) the last frame (14.16${\mathrm{e}}^{-}/{\mathrm{\AA }}^2$). b)–d) show the fits to simulated data (superimposed strip at lower half) using the near equipotential surface charge distribution and the sample charge used for the simulation.

Fig. 2

Charge distribution models and their associated electrical potential distributions. a) Two charge distribution models of an equipotential surface and a uniform charge surface. The charge distributions are plotted for one $q_e=1.602\times {10}^{-19}$ C charge in the illuminated area. b) Phase profiles calculated for electrons passing through the three charge distributions. Fits to ideal lenses and ideal lenses with third order spherical aberration are shown. The vertical dotted lines represent the boundaries of the illuminating electron beam. The uniform and equipotential charge distributions are calculated for one $q_e$ charge. The screened charge distribution is calculated for 1350 $q_e$ charge. c) Surface charge distribution model due to secondary electron emission. Plots for different percentages of secondary electrons effectively lost to vacuum are shown. d) Contour plot of potential distribution around a uniform surface charge distribution with one $q_e$ charge. e) Contour plot of the potential distribution around a equipotential surface charge distribution with one $q_e$ charge. f) Contour plot of the potential distribution around a screened uniform surface charge distribution with 1350 $q_e$ charge.

Fig. 3

a-c) Plots of the diffraction disc beam radius as a function of accumulated dose as measured from Fresnel diffraction movies recorded at 40 fps. a) Various ice films irradiated with 1.51${\mathrm{e}}^{-}/{\mathrm{\AA }}^2/\mathrm{s}$, b) various ice films irradiated with 0.36${\mathrm{e}}^{-}/{\mathrm{\AA }}^2/\mathrm{s}$, c) carbon film irradiated with 1.51${\mathrm{e}}^{-}/{\mathrm{\AA }}^2/\mathrm{s}$ (blue line) and illuminated ice films with the surrounding carbon film moved into the illumination area by varying amounts. d) (left axis) The average electron dose at which the diffraction disc radius drops below a threshold value defined as $1/e^2$ of the normalized disc radius for various dose-rates. (right axis) The average charge on the samples as determined by the charge value which gave the best fit to frames in the semi-stable regions for various dose-rates. The error bars represent the standard deviation between 4 samples under the same dose-rate conditions.

Fig. 4

a) Fitted charge Q (left y-axis) and propagation distance $z_p$ (right y-axis) values for an ice film sample exposed to a 1.51${\mathrm{e}}^{-}/{\mathrm{\AA }}^2/\mathrm{s}$ dose-rate. The double-slash on the x-axis represents a jump in the accumulated dose scale markings in order to make the behavior at low accumulated dose more visible. b) Fitted charge and propagation distance values for an ice film sample with the surrounding carbon film entering inside the beam area imaged with a 0.48${\mathrm{e}}^{-}/{\mathrm{\AA }}^2/\mathrm{s}$ dose-rate.

Fig. 5

a) Frame of the central diffraction disc when the electron beam is 72 nm away from the edge of the carbon film (represented by the dotted curve). b) Sum of frames of the central diffraction disc when a carbon film is 32 nm inside the area illuminated by the electron beam. The darker area is the carbon film. Red dotted and blue dashed circles show the radius associated with carbon and ice areas. Simulated Fresnel patterns for the carbon and ice areas and their associated charge values are also shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)