Vitrifying Cells: it takes Two to Tango

Structural and functional knowledge of proteins, which are essential in biological processes, is fundamental for our understanding of the Chemistry of Life. Structural biology - the field that studies the structure and function of proteins – has seen several revolutions over the last few years. Single particle analysis (SPA), where individual macromolecular assemblies are imaged under cryogenic conditions within highly automated electron microscopes, has been used to elucidate the structures of many novel and important proteins and complexes. Deep-learning–based computational techniques provided systematic predictions of an million three-dimensional protein structures. Cryo-electron tomography (ET) combined with sub-tomogram averaging (STA) enabled the investigation of conformational states of large macromolecular complexes. We expect in situ structural biology, where macromolecular assemblies are studied within the interior of focused-ion-beam milled frozen cells, to become the next revolution in our field. Such revolution would require well prepared vitreous samples (cells, tissue slices, organoids): the sample should be cooled fast enough to prevent the formation of crystalline ice. Previously, we developed the technology to prepare SPA samples using jets of cryogenic fluid directed onto the sample. This device, the VitroJet, has been further developed into a commercial product by CryoSol-World and has been sold worldwide. Here, we wish to advance the jetting technology such that it can vitrify cells. Crucial aspects are the speed of the jets and the timing and reproducibility of the fronts of the cryogens arriving onto the sample. We will design, build, characterise and refine a next generation of the ethane cup, a core component within the VitroJet. If successful, we should be able to increase its vitrification potential as well as its reproducibility by more than one order of magnitude. This technology will enable in situ structural biology studies necessary to understand the Chemistry of Life.