Stroboscopic image capture: Reducing the dose per frame by a factor of 30 does not prevent beam-induced specimen movement in paraffin

Abstract

Beam-induced specimen movement may be the major factor that limits the quality of highresolution images of organic specimens. One of the possible measures to improve the situation that was proposed by Henderson and Glaeser (Henderson and Glaeser, 1985), which we refer to here as "stroboscopic image capture", is to divide the normal exposure into many successive frames, thus reducing the amount of electron exposure -and possibly the amount of beaminduced movement -per frame. The frames would then be aligned and summed. We have performed preliminary experiments on stroboscopic imaging using a 200-kV electron microscope that was equipped with a high dynamic range CCD camera for image recording and a liquid N 2 -cooled cryoholder. Single-layer paraffin crystals on carbon film were used as a test specimen. The ratio F(g)/F(0) of paraffin reflections, calculated from the images, serves as our criterion for the image quality. In the series that were evaluated, no significant improvement of the F image (g)/F image (0) ratio was found, even though the electron exposure per frame was reduced by a factor of 30. A frame-to-frame analysis of image distortions showed that considerable beam-induced movement had still occurred during each frame. In addition, the paraffin crystal lattice was observed to move relative to the supporting carbon film, a fact that cannot be explained as being an electron-optical effect caused by specimen charging. We conclude that a significant further reduction of the dose per frame (than was possible with this CCD detector) will be needed in order to test whether the frame-to-frame changes ultimately become small enough for stroboscopic image capture to show its potential.

A dose-rate effect in single-particle electron microscopy

“…The volume filled by a free radical is larger than that of a radical covalently bound to a molecule, because the distance between radicals and surrounding atoms is dictated by the van der Waals radii (>3 Å), which is larger than the length of a covalent bond (~1.5 Å). The formation of radicals is therefore associated with the buildup of pressure inside the specimen [Typke et al, 2007]. For a high-intensity beam, the pressure can become so high that it generates mechanical fractures within the specimen, most likely at mechanically weakened interfaces between ice and carbon film, and between ice and protein.…”

“…The multiple frames are then coherently summed to form a single, SNR-enhanced image for analysis. A similar scheme has also been proposed by Typke et al, 2007. The image alignment can be achieved, either through a correlation-based approach or via a fiducial-marker registration.…”

Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM

“…In practice, beam induced motion seems impossible to prevent 14,15 , and deteriorates most images from “perfect” to marginal for high-resolution cryoEM 16 . The effect can be approximated as Gaussian blurring and quantified by analogy with the crystallographic temperature factor 17–19 , predicting a greater than 5-fold decrease in signal at 3 Å 13 .…”

“…That is, a single exposure is fractionated into a number of subframes with sufficiently short duration that the motion is frozen to an acceptable level 13 . The drawback is that while the signal decreases with fractionation, camera readout noise on each subframe remains constant 15 . Even so, in the case of large objects such as viruses 20,21 or ribosomes 22 particles, it has been possible to use the subframes as if they were independent images during the refinement resulting in an increase in resolution.…”

Precise beam-tilt alignment and collimation are required to minimize the phase error associated with coma in high-resolution cryo-EM

“…One must then apply a systematic correction to the higher-frequency Fourier components of the image in order to compensate for the resulting oscillations in the phase-contrast transfer function (CTF), sin γ ( s ), where γ ( s ) is the amount by which the phase of the electron wave-front at the back focal plane of the objective lens deviates relative to the phase of a converging spherical wave. One must also take account of an overall envelope of the contrast transfer function that occurs due to beam-induced movement (Henderson and Glaeser, 1985; Typke et al, 2007), partial spatial and temporal coherence (Frank, 1973; Frank, 2006; Wade and Frank, 1977), and the modulation transfer function (MTF) of the detector. For simplicity, we do not consider the envelope of the CTF in this review; suffice it to say that one can compensate for the envelope by empirical sharpening of the density map (Fernandez et al, 2008).…”