Defocus and magnification dependent variation of TEM image astigmatism.

Daily alignment of the microscope is a prerequisite to reaching optimal lens conditions for high resolution imaging in cryo-EM. In this study, we have investigated how image astigmatism varies with the imaging conditions (e.g. defocus, magnification). We have found that the large change of defocus/magnification between visual correction of astigmatism and subsequent data collection tasks, or during data collection, will inevitably result in undesirable astigmatism in the final images. The dependence of astigmatism on the imaging conditions varies significantly from time to time, so that it cannot be reliably compensated by pre-calibration of the microscope. Based on these findings, we recommend that the same magnification and the median defocus of the intended defocus range for final data collection are used in the objective lens astigmatism correction task during microscope alignment and in the focus mode of the iterative low-dose imaging. It is also desirable to develop a fast, accurate method that can perform dynamic correction of the astigmatism for different intended defocuses during automated imaging. Our findings also suggest that the slope of astigmatism changes caused by varying defocuses can be used as a convenient measurement of objective lens rotation symmetry and potentially an acceptance test of new electron microscopes.