High-quality, high-throughput cryo-electron microscopy data collection via beam tilt and astigmatism-free beam-image shift.

The throughput of cryo-electron microscopy (cryo-EM) can be improved by employing a procedure that collects beam-image shift data. However, this procedure inadvertently induces a beam tilt, thus decreasing the resolution of the reconstruction. Here, we report an automatic calibration procedure for correcting the beam tilt and a large unexpected astigmatism in the beam-image shift data collection. In this procedure, the changes of the beam tilt and the astigmatism against the beam shift are measured and calibrated. The beam tilt and the astigmatism are corrected by changing the setting of the microscope using predicted values from the calibration. Using our corrected beam-image shift data collection, we found that the resolution remained identical as long as the distance of the beam shift was below 10 μm. The image throughput increases by ∼80%, with image quality improving by reducing the residual stage drift, thus benefiting the high resolution cryo-EM structure determination. Such a calibration procedure takes about 3 h and can be applied to different microscopes.