The recent technological breakthrough in cryo electron tomography (cryoET) opens to imaging, at close to atomic resolution, of macromolecular complexes in their native cellular context. This technique is seminal for a new field in Life Sciences referred to as cellular structural biology which is starting to shed light on how fine conformational states of protein complexes are linked to biological functions. In parallel, molecular cell biology is evolving towards integrated systems, where fundamental cellular functions are studied at multiple dimensions of space and time in model organisms. Whilst cellular structural biology is now routinely performed on simplified single cell models, it is still extremely challenging on organisms, mostly due to technical hurdles linked to the huge scale range that one has to cover for switching from millimetre-scale organisms or tissues to the nanometre-range objects imaged by cryoET. For doing so, objects of interest have to be selected and targeted in three dimensions, then physically extracted for high resolution imaging. Correlative Light and Electron Microscopy (CLEM) is one way to combine functional and structural imaging on the same samples. For multicellular models, X-ray Computed Tomography (CT) has a crucial role to play in such combined workflows as it bridges the resolution gap and enables precise
targeting of key features within the tissues.

By synergising three independent projects within the framework of this Attract Open Call, we propose to tackle three key steps in such targeting workflow. The first project, and the one described herein, UTXμCT deals with the ability to build precise three-dimensional (3D) maps of vitrified specimens by phase contrast computed X-ray tomography.

A second aim mIR-ivSEM is to develop algorithms and software to load such 3D maps onto scanning electron microscopes, dedicated to the preparation of thin lamellas from vitrified specimens.

The third project MSET- will tackle the challenge of precisely targeting, creating and manipulating 100 nm-scale lamellae for their characterisation by cryo-ET, aided by cryo focused ion beam (FIB)/ scanning electron microscopy (SEM) tomography.

UTXμCT aims at the demonstration of three-dimensional imaging of unstained biological samples at better than one micron isotropic resolution, with a bench-top X-ray phase-contrast imaging system. Precise targeting, and subsequent preparation of thin lamellas for cryo electron tomography, in pristinely preserved tissues and organisms will be enabled by removing the need of heavy metal staining in the sample preparation. This multi-scale imaging workflow reaches close to atomic resolution, in its final stage, while preserving the contextual information of the whole organisms and tissues (millimetre-scale) from which the thin lamellas are extracted. We envision that developing this multi-scale imaging workflow, from millimetre-size organisms and tissues, down to below the nanometre with complementary cryo- ET approaches, will lend an enormous discovery potential to any life, biomedical and material science study worldwide.