2024

Henderikx, Rene J. M.; Mann, Daniel; Domanska, Aušra; Dong, Jing; Shahzad, Saba; Lak, Behnam; Filopoulou, Aikaterini; Ludig, Damian; Grininger, Martin; Momoh, Jeffrey; Laanto, Elina; Oksanen, Hanna M.; Bisikalo, Kyrylo; Williams, Pamela A.; Butcher, Sarah J.; Peters, Peter J.; Beulen, Bart W. A. M. M.

Single-particle cryo-electron microscopy has become a widely adopted method in structural biology due to many recent technological advances in microscopes, detectors and image processing. Before being able to inspect a biological sample in an electron microscope, it needs to be deposited in a thin layer on a grid and rapidly frozen. The VitroJet was designed with this aim, as well as avoiding the delicate manual handling and transfer steps that occur during the conventional grid-preparation process. Since its creation, numerous technical developments have resulted in a device that is now widely utilized in multiple laboratories worldwide. It features plasma treatment, low-volume sample deposition through pin printing, optical ice-thickness measurement and cryofixation of pre-clipped Autogrids through jet vitrification. This paper presents recent technical improvements to the VitroJet and the benefits that it brings to the cryo-EM workflow. A wide variety of applications are shown: membrane proteins, nucleosomes, fatty-acid synthase, Tobacco mosaic virus, lipid nanoparticles, tickborne encephalitis viruses and bacteriophages. These case studies illustrate the advancement of the VitroJet into an instrument that enables accurate control and reproducibility, demonstrating its suitability for time-efficient cryo-EM structure determination.