A versatile focused ion beam milling microscope for in situ structural biology

The Nobel prize-winning technology of cryogenic Electron Microscopy (cryoEM) has transformed structural biology research, furthering our knowledge of biology, providing novel insights into the molecular mechanisms of health and disease, enabling drug design, and driving engineering biology efforts. Until recently, high-resolution cryoEM was limited to purified proteins and complexes, which necessitates removing the protein from its native environment. We therefore lose in situ information, which contains the functional data about the cellular context. Cryogenic electron tomography (cryoET) provides this information, but samples must be less than 200 nm thick for high-resolution imaging, whereas mammalian cells are >5000 nm thick, which completely precludes imaging. To produce thin samples, the optimal method is focussed ion beam (FIB) milling, performed at cryogenic temperatures (-180°C). Such a cryo-FIB removes material with nanometre precision, leaving behind a lamella - a thin slice - through the sample (e.g., cell, tissue, biopsy, etc). Cryo-FIB-SEMs contain integrated fluorescence modules that allow for targeted milling towards the fluorescent regions/molecules of interest, making the process more efficient and time-saving.

CryoEM in the south-west of the UK is world-leading and highly collaborative, supported by the GW4 facility for high-resolution cryoEM. It is used extensively by the Universities of Bath, Bristol, Cardiff and Exeter (GW4) and beyond. Since 2017, the facility has enabled the determination of dozens of structures. However, research has so far largely focussed on single-particle analysis approaches. Driven by the aspiration and need of GW4 researchers to incorporate in situ structural biology using cryoET, the Universities of Bristol (the host institute) and Exeter have recently invested in equipment and personnel to expand the region's state-of-the-art cryoEM capabilities. In particular, the recent recruitment of Thom Sharp, a cryoET specialist, to Bristol was borne with that vision in mind. We seek to acquire the first cryo-FIB-SEM in the south-west of UK dedicated to strengthen in situ structural biology research in the region.

Various types of integrated fluorescence cryo-FIB-SEMs are available; some are designed for the one single task of lamella preparation, which limits the application spectrum of such an (expensive) instrument. Here, we are applying for a microscope that, in addition to the targeted lamella preparation, will allow for "routine" cryo-SEM applications and uniquely incorporate elemental analysis capabilities (EDS) under cryo-conditions without compromising the cryo lamella preparation capabilities.

Such an instrument would provide completely novel capabilities for GW4 but importantly will also replace an over 18-year old cryo-SEM and integrate the new capabilities (targeted lamella preparation and cryo-EDS) with existing ones ("routine" cryo-SEM) into 1 instrument to increase sustainability rather than running 2 individual instruments.

The new instrument will support a very wide range of research projects, and herein we demonstrate the need for these new capabilities by 4 detailed case studies from GW4 researchers supplemented with the titles of an additional 18 planned projects, addressing fundamental biological understanding, advancing cell biology for an integrated understanding of health, engineering biology, and technology development, all key BBSRC strategic areas.