Optimized data acquisition workflow by sample thickness determination

Sample thickness is a known key parameter in cryo-electron microscopy (cryo-EM) and can affect the amount of high-resolution information retained in the image. Yet, common data acquisition approaches in single particle cryo-EM do not take it into account. Here, we demonstrate how the sample thickness can be determined before data acquisition, allowing to identify optimal regions and restrict automated data collection to images with preserved high-resolution details. This quality over quantity approach, almost entirely eliminates the time- and storage-consuming collection of suboptimal images, which are discarded after a recorded session or during early image processing due to lack of high-resolution information. It maximizes data collection efficiency and lowers the electron microscopy time required per dataset. This strategy is especially useful, if the speed of data collection is restricted by the microscope hardware and software, or if microscope access time, data transfer, data storage and computational power are a bottleneck. SynopsisSample thickness is a key parameter in single particle cryo-electron microscopy. Determining sample thickness before data acquisition allows to target optimal areas and maximize data output quality of single particle cryo-electron microscopy sessions. Scripts and optimized workflows for EPU and SerialEM are presented and available as open-source.