VitroJet: next generation sample preparation for cryo-EM

B.W.A.M.M. Beulen1, R.J.M. Henderikx1, G. Weissenberger1, F.J.T. Nijpels1, C. Lopez-Iglesias2, R.B.G. Ravelli2, P.J. Peters2

1CryoSol-World (www.cryosol-world.com), the Netherlands 2Maastricht University, the Netherlands

bart.beulen@cryosol-world.com

Through many exciting developments, cryo-electron microscopy (EM) has emerged as a powerful technique to elucidate the 3D structure of macromolecular complexes. This knowledge provides valuable insight in understanding their functions and is the foundation for developing novel medicines.

Sample preparation is an essential step in cryo-EM: for studying macromolecular in their native state, a specimen needs to be cooled so rapidly that ice crystals are unable to form and cause damage. Trapping the molecules in amorphous ice is called vitrification. Traditionally, vitrification was performed by manually plunging the sample on an EM-grid in liquid ethane. In the late 1990s the first automated plunge-vitrification instruments were developed.

With the increasing demand on cryo-EM, sample preparation has become the major bottleneck that limits the true potential of cryo-EM. Current sample preparation solutions still rely on the methods established in the 1980s which are limited with respect to efficiency and reproducibility. Though automated cryo-EM microscopes are common nowadays, plunge vitrification does not offer sufficient cooling capacity to cope with the added thermal mass of the sturdy ring-shaped in which EM-grids need to be mounted to enable the automated handling. Consequently, mounting the fragile EM-grid in the ring-shaped support needs to be performed manually under cryogenic conditions post vitrification, severely limiting throughput and further reducing reproducibility.

We developed the VitroJet[1], a fully automated sample preparation solution for cryo-EM in which after highly reproducible thin layer deposition of the sample onto the EM-grid, two simultaneous jets of liquid ethane are targeted onto the center of the EM-grid[2]. This raises cooling capacity, and results in increased cooling rates that ensure successful vitrification of samples on EM-grids that are already mounted in the ring-shaped support. Consequently, EM-grids do not need to be post-mounted anymore, further improving reproducibility of the results. The automated approach enables users to efficiently optimize sample conditions in the race to obtain new and exciting macromolecular structures.

[1] “Automated cryo-EM sample preparation by pin-printing and jet vitrification” BioRxiv, https://doi.org/10.1101/651208

[2] Patents pending