28 29 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 Biological Cryo-Imaging Group MartinWalsh, Science Group Leader T he Biological Cryo-imaging Group brings together dedicated facilities for X-ray, light and electronmicroscopy at Diamond Light Source. The electron BioImaging Centre (eBIC) is the national centre for cryo-Electron Microscopy (cryo-EM) in the UK and provides a range of capabilities and supporting facilities for cryo-EM and Correlative Light and Electron Microscopy (CLEM). Beamline B24 hosts a full field cryo-transmission X-ray microscope dedicated to biological X-ray imaging and has also established a cryo super resolution fluorescence microscopy facility, which is a joint venture betweenDiamond and the University of Oxford. It provides a unique platformfor correlative light and X-ray microscopy, and cryo-EM. The Biological Cryo-Imaging group continues to grow and welcome new faces as we finalise our recruitment drive at eBIC and renew posts at B24. At eBIC two cryo-EM scientists Matt Bryne and Lorna Malone and new PhD students Emily Lacey, Frank Nightingale and Patrick Phillips, and a year in industry student (Ishika Kumar) have recently joined. In addition, the Post- Doctoral research team has expanded with the addition of two new researchers Kyprianos Hadjidemetriou and Satinder Kaur, who are funded on a new strategic ERC grant awarded to the eBIC director Peijun Zhang. At B24, Chidinma Okolo and Archana Jadhav have been appointed as beamline scientists. Last autumn B24 underwent its first external beamline review which independently assessed the performance of the beamline to date and rated it overall as excellent and world-leading. In particular, the external panel commended the beamline team on establishing an internationally unique correlative platform combining two high-end 3D cryo-microscopy techniques (Cryo Soft X-ray Tomography (CryoSXT) and cryo Structured Illumination Microscopy (SIM)) alongside a user friendly package of protocols that encompass sample preparation methods and in silico procedures for data correlation and analysis. As a result of the pandemic and with users, until recently, not being able to attend site, we continue to evolve our remote user activites and continue with developing remote training/teaching courses and material. At eBIC we have rolled out regular remote user training courses for our Block Allocation Groups and extensive new user guides for eBIC microscopes. Similarly at B24 the team has publishedmanuals and practical guidelines for users on their webpages. B24 has also worked hard to disseminate beamline capabilities from the basics to correlative imaging through workshops ( e.g . iNEXT-Discovery workshop “ What can Cryo Electron Tomography and Cryo Soft X-ray Tomography do? ” ) as well as publish beamline protocols and methodologies in peer-reviewed publications. Highlights from eBIC's internal research programme includes the release and corresponding publication of a new software tool, IceBreaker, that helps in optimisation of particle picking and data collection 1 . The method has been developed by a joint PhD student (Mateusz Olek) between Diamond and the University of York. The aim of the method is to resolve adverse effects of variable ice thickness in cryo-EM. It consists of image segmentation and statistical analysis of the background, followed by discrete normalisation of patches. This allowed for better identification of particles irrespective of the background originating from the ice. This impacts and aids researchers to now reliably gather more structural information by analysing more protein particles. IceBreaker is now fully integrated in the eBIC single particle image processing pipeline. Publication highlights for B24 alongside a number of instructional/ educational and review articles, include a paper in Nature Protocols which documents all steps necessary for successful sample preparation for Soft X-ray Tomography 2 , work on SARS-Cov2 in partnership with eBIC and the University of Oxford 3 and further work on novel T cell-derived cytotoxic particles 4 . Finally, at B24 we have implemented automated data collection from our transmission X-ray microscope which plugs neatly into our already existing automated data reconstruction pipeline. The partnership between Diamond and the Research Complex at Harwell (RCaH) has been strengthened by the recruitment of Michal Klosowski – who will manage the RCaH 200 keV Thermofisher Scientific Glacios electron microscope housed at eBIC. The RCaHGlacios is also currently being used to asses the Dectris SINGLA detector, a 1 Mega-pixel, hybrid pixel electron detector for micro-crystal Electron Diffraction (microED). This is carried out in collaboration with the Diamond HeXI beamline team and CCP4 where the Dials software package is being integrated to provide a data processing pipeline for the data generated. New capabilities on the horizon at eBIC include new access routes for using the Leica cryo Correlative Light and Electron Microscope (cryoCLEM) as well as access for microED. Coupled to this, eBIC has continued working extensively with the manufacturer Thermofisher to commission and benchmark their Selectris-X energy filter and updated Falcon 4i detectors which were brought into the user program at the start of 2022. References 1. Olek, M. et al. IceBreaker: Software for high-resolution single-particle cryo- EMwith non-uniform ice. Structure 30 , 522-531.e4 (2022). DOI: 10.1016/j. str.2022.01.005 2. Okolo, C. A. et al. Sample preparation strategies for efficient correlation of 3D SIM and soft X-ray tomography data at cryogenic temperatures. Nature Protocols 16 , 2851–2885 (2021). DOI: 10.1038/s41596-021-00522-4 3. Mendonça, L. et al. Correlative multi-scale cryo-imaging unveils SARS- CoV-2 assembly and egress. Nature Communications 12 , 4629 (2021). DOI: 10.1038/s41467-021-24887-y 4. Chang, H.-F. et al. Identification of distinct cytotoxic granules as the origin of supramolecular attack particles in T lymphocytes. Nature Communications 13 , 1029 (2022). DOI: 10.1038/s41467-022-28596-y Highlights fromeBICs internal research program includes the release and corresponding publication of a new software tool, IceBreaker, that helps in optimisation of particle picking and data collection. D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 The eBIC team .