The progressive spectral signal-to-noise ratio of cryo-electron micrograph movies as a tool to assess quality and radiation damage

Abstract

Background and ObjectiveThe quality of a cryoEM reconstruction is fundamentally a function of the signal-to-noise ratio (SNR) of the original micrographs. The SNR embodies multiple aspects of image formation, including microscope details and alignment, specimen composition and thickness, how it is recorded, and how the specimen degrades during imaging. With the advent of direct electron detectors and the recording of a series of images for each micrograph (a movie), we have an opportunity to count every electron and derive fully quantitative results. After alignment of the movie frames of a micrograph, we can calculate the SNR, or its spatial frequency equivalent, the spectral SNR (SSNR). This SSNR reflects residual movement between frames and the progressive effect of radiation damage. The goal is to develop a quantitative analysis of the SSNR and radiation damage to assess and improve the quality of micrographs. MethodsSeveral test cases were selected from the EMPIAR database and ten micrograph movies downloaded for each case. The movie frames were aligned as rigid bodies to compensate for stage and support movement. The SSNR for subsets of frames was then calculated to assess the effect of residual movement. The progressive SSNR (PSSNR) was subsequently calculated to determine the decrease in signal accumulation as a result of radiation damage. ResultsIn all cases the alignment of the movie frames compensated for global movement to the extent that the effect on the SSNR is negligible. The subset SSNR can be used as a tool to further confirm the extent of residual movement. The progressive SSNR indicates an increase in value up to an asymptote, consistent with the theory for radiation damage. Fitting these curves gives the inherent SNR before exposure, and the critical dose, which decreases with spatial frequency with an exponential parameter roughly between one and two. ConclusionsThe implementation of the PSSNR for movie frames provides a tool for assessing micrograph quality and progression of radiation damage. The estimation of the critical dose further quantifies radiation damage and may shed some light on the mechanisms of damage. These are likely both a function of the specimen composition and the imaging parameters used.