Abstract
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.
Highlights
Cryo-electron microscopy has become a powerful technique for structural studies of macromolecular complexes and assemblies at near-atomic resolutions
Astigmatism of the objective lens represents the angular dependency of defocus. 2-fold astigmatism is the major type of astigmatism relevant to Cryo-electron microscopy (cryo-EM), which results in the elliptical elongation of Thon rings[3] in the power spectra of TEM images
It is worth pointing out that this correlation is a general phenomenon since it is observed in a wide variety of data, such as data from multiple research groups, microscopes in different facilities, a diversity of samples, and varying imaging conditions
Summary
Cryo-electron microscopy (cryo-EM) has become a powerful technique for structural studies of macromolecular complexes and assemblies at near-atomic resolutions. Due to the poor sensitivity of human eyes, microscopists have to rely on large magnifications and small defocuses when they visually examine the roundness of Thon rings in the 2D power spectra displayed on a computer screen and iteratively adjust the two objective lens stigmators (we use MX and MY to call the two stigmators as they are natural abbreviations of the Multi-functional knobs X and Y used to adjust the current of the two stigmators) to make the Thon rings as circular as possible This tedious and subjective method is inaccurate www.nature.com/scientificreports/. We have recently published a method, s2stigmator[9], with a single-pass tuning strategy, that allows rapid and sensitive detection of astigmatism using TEM live images and can reliably and efficiently guide the user to manually adjust the two stigmators to correct astigmatism This new method opens up possibilities to minimize astigmatism with real-time feedback at a wide range of imaging conditions that are not available by visual examination. Based on the findings of these studies, several recommendations are provided for instrument alignment and data acquisition to help maximally reduce astigmatism and improve high resolution imaging
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