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The effects of dose and dose-rate were investigated for single-particle cryo-electron microscopy using stroboscopic data collection. A dose-rate effect was observed favoring lower flux densities.

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Charge-coupled devices (CCD) are nowadays commonly utilized in transmission electron microscopy (TEM) for applications in life sciences. Direct access to digitized images has revolutionized the use of electron microscopy, sparking developments such as automated collection of tomographic data, focal series, random conical tilt pairs and ultralarge single-particle data sets. Nevertheless, for ultrahigh-resolution work photographic plates are often still preferred. In the ideal case, the quality of the recorded image of a vitrified biological sample would solely be determined by the counting statistics of the limited electron dose the sample can withstand before beam-induced alterations dominate. Unfortunately, the image is degraded by the non-ideal point-spread function of the detector, as a result of a scintillator coupled by fibre optics to a CCD, and the addition of several inherent noise components. Different detector manufacturers provide different types of figures of merit when advertising the quality of their detector. It is hard for most laboratories to verify whether all of the anticipated specifications are met. In this report, a set of algorithms is presented to characterize on-axis slow-scan large-area CCD-based TEM detectors. These tools have been added to a publicly available image-processing toolbox for MATLAB. Three in-house CCD cameras were carefully characterized, yielding, among others, statistics for hot and bad pixels, the modulation transfer function, the conversion factor, the effective gain and the detective quantum efficiency. These statistics will aid data-collection strategy programs and provide prior information for quantitative imaging. The relative performance of the characterized detectors is discussed and a comparison is made with similar detectors that are used in the field of X-ray crystallography.

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Image formation in phase-contrast electron microscopy is governed by the contrast transfer function (CTF). The key parameter to tune the CTF is the defocus. A precise and unbiased estimate of the defocus is essential to determine the forward model and interpret high resolution images in cryo-TEM. We present an algorithm based on the weak-phase approximation for determination of the defocus and astigmatism of the objective lens from recorded images of an amorphous sample. The algorithm identifies Thon rings in the power spectrum density (PSD) and uses them to estimate astigmatism and defocus together with their uncertainties. For the astigmatism estimation we use a transformation to polar coordinates to fit ellipses to the Thon rings. Averaging the PSD over these ellipses reduces CTF estimation to a 1D problem and enhances the signal-to-noise ratio.

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