Automated vitrification of cryo-EM samples with controllable sample thickness using suction and real-time optical inspection

Abstract

The speed and efficiency of data collection and image processing in cryo-electron microscopy have increased over the last decade. However, cryo specimen preparation techniques have lagged and faster, more reproducible specimen preparation devices are needed. Here, we present a vitrification device with highly automated sample handling, requiring only limited user interaction. Moreover, the device allows inspection of thin films using light microscopy, since the excess liquid is removed through suction by tubes, not blotting paper. In combination with dew-point control, this enables thin film preparation in a controlled and reproducible manner. The advantage is that the quality of the prepared cryo specimen is characterized before electron microscopy data acquisition. The practicality and performance of the device are illustrated with experimental results obtained by vitrification of protein suspensions, lipid vesicles, bacterial and human cells, followed by imaged using single particle analysis, cryo-electron tomography, and cryo correlated light and electron microscopy.

Faster cryo specimen preparation can advance cryo electron microscopy (cryoEM). Here, the authors present a vitrification device with automated sample handling for cryoEM of proteins, suspensions and cells, enabling blot-free sample thinning, dew-point control and characterization of cryo grids prior to data acquisition.