IG17121 - Faites Vos Jeux! Moving high-resolution in cellulo structural biological of infectious processes from the roulette table to the chess board

After the Nobel prize in chemistry for cryo-electron microscopy (cryoEM) in 2017, the field has been growing exponentially with many/all structural biologists adopting now cryo-Transmission Electron Microscopy (cryo-TEM) Single Particle Analysis (SPA) as a standard method to resolve protein structures at molecular resolution. Cell biologist have been using TEM on dehydrated, plastic embedded biological samples for decades as the only method to discover the structural features in the cell. Now, a new method has been developed to cut TEM-transparent windows in a vitrified cell (Cryo focused ion beam, cryo-FIB). Cell biologists can now obtain a view of the true native – unstained, unfixed and fully hydrated – state of the cell with a similar revolutionary clarity as has been possible for the structural biologists while profiting of the latest breakthrough camera development. Since the onset of cryo-FIB, researchers have been adapting standard, decades old, EM-grids to do their experiments. Sample carriers (EM-grids) however did not benefit from these novel advances and are still based on outdated standard models. These grids were never made, nor designed with this new technologies and potential in mind. Cells are currently cultured randomly on EM grids and randomly cut and imaged without any previous knowledge about their state. The imaging, sometimes assisted by cryo-fluorescence microscopy (cryo-CLEM), is generally performed in a brute force method with hopes to find structures of interest within large data sets. We propose to develop a new EM-grid and workflow that will revolutionize the cryo-FIB/cryo-CLEM field. We plan to design less fragile grids to deposit, culture and image single cells of know biological state, similarly to creating a “chess board”. The regular design of our functionalized EM-grids will also allow for rational highthroughput screening, resulting in comprehensive data sets on single grids. We predict that the high regularity of the grid will also, for the first time, provide the means for full automation and thus massive simplification of the existing workflows relying on cryo-FIB milling of lamella followed by automated tomography, thus removing the element of chance from the equation. We strive to develop the next standard sample carrier for cryo-TEM cellular biology for the coming decades.