In cryo-electron microscopy structure determination, embedding biomolecules in vitreous ice of optimal thickness is critical. Ice thickness assessment and selection of suitable holes for data collection are currently part of time-consuming preparatory routines performed on expensive electron microscopes. For this reason, we developed a method to determine ice thickness during sample preparation using the optical camera integrated in the VitroJet. As a result, the ice thickness of buffer-suspended holes on an EM grid can be determined faithfully in the working range relevant for single particle cryo-EM. Dependent on the thickness, the error is below +- 20 nm in the range between 0 - 70 nm down to +- 10 nm in the thinnest ice regions (10 - 40 nm). Single particle structures of apoferritin were determined at two distinct thicknesses of 30 nm and 70 nm to demonstrate the suitability of optical layer thickness measurements for time-efficient cryo-EM structure determination.

Optical ice thickness measurement in the VitroJet for time-efficient single particle structure determination