A dose-rate effect in single-particle electron microscopy

Abstract

A low beam intensity, low electron dose imaging method has been developed for single-particle electron cryo-microscopy (cryo-EM). Experiments indicate that the new technique can reduce beam-induced specimen movement and secondary radiolytic effects, such as "bubbling". The improvement in image quality, especially for multiple-exposure data collection, will help single-particle cryo-EM to reach higher resolution.

Figure 1

Two consecutive exposures of colloidal gold fiducial markers (10 nm, dark dots) in vitreous ice. The intra-particle distance change between two exposures is defined as (d1–d2).

Figure 2

a) Intra-marker distance change between the double exposures. The beam-induced particle movement is larger in HiFlux. Both distributions are skewed towards the positive side, which indicates an overall contraction of the specimen. b) Absolute intra-marker distance change correlates with the marker-pair separation. The beam-induced particle movement is much smaller in LINDA. c) Continuous particle movement during multiple exposures. The lines represent least-square fitting (y = kx) to the respective datasets. Using the triplet (k, Δk, ε)_n to designate the slope k, the error in k and the fitting variance from the n-th exposure, the results are (0.81,0.19,3.65)₂, (0.99,0.23,3.97)₃, (1.41,0.35,5.06)₄ and (1.82,0.47,7.34)₅.

Figure 3

HiFlux (top) and LINDA (bottom) imaging. Three consecutive exposures by each protocol. All images are collected at 200keV, 3.5 μm defocus on an FEI TF30 electron microscope. The ice droplets in A1 is noticeably smaller than those in B1. Since these images are collected in the same area on the specimen grid, the ice droplets are expected to have similar size before beam exposure. The smaller size in A1 is due to a stronger sublimation from the HiFlux beam exposure.

Figure 4

Comparing specimen bubbling under HiFlux and LINDA imaging modes. Bubbling occurs at significantly lower total dose under HiFlux conditions.

Figure 5

a) Image power-spectrum comparison between LINDA and HiFlux. The peaks (labeled by A, B, C, D) from LINDA are stronger and extend further to higher resolution (the arrows). b) Image power spectrum comparison between LINDA and HiFlux imaging methods. The defocus of both images is around 2.5 μm in this example. The beam envelope function, as judged from the Thon ring amplitudes, is comparable in the resolution range of interest.

Figure 6

A TMV 3D density map reconstructed from LINDA imaging. Left: a side view. Right: a central section.