Energy, Materials & Systems (EMS)

Automated cryo-EM sample preparation by pin-printing and jet vitrification

Raimond B.G. Ravelli1*, Frank Nijpels1,2, Rene J.M. Henderikx1,2, Giulia Weissenberger1,2, Sanne Thewessem3, Abril Gijsbers1, Bart Beulen2,3, Carmen López-Iglesias1, Peter J. Peters1,2*

1The Maastricht Multimodal Molecular Imaging Institute (M4I), Division of Nanoscopy, Maastricht University, Maastricht, the Netherlands; 2CryoSol World, Maastricht, the Netherlands; 3Instrument Development, Engineering and Evaluation (IDEE), Maastricht University, Maastricht, the Netherlands

*Corresponding authors:;

The increasing demand in cryo-electron microscopy (cryo-EM) reveals drawbacks in current sample preparation protocols, such as sample waste and lack of reproducibility. Here, we present several technical developments that provide controlled and efficient sample preparation for cryo-EM studies. A sub-nanoliter volume of sample is deposited on the carrier surface by pin printing. Sample evaporation is mitigated by dewpoint control feedback loops. The deposited sample is vitrified by jets of cryogen followed by submersion into a cryogen bath. Because the cryogen jets cool the sample from the center, premounted autogrids can be used and loaded directly into automated cryo-EMs. We integrated these steps into a single device, named VitroJet. The device's performance was validated by resolving 4 standard proteins (apoferritin, GroEL, worm hemoglobin, beta-galactosidase) to ~3 Å resolution using a 200-kV electron microscope. The VitroJet offers a promising solution for improved automated sample preparation in cryo-EM studies.