Diamond Light Source - Annual Review 2022/23

18 19 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 2 / 2 3 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 2 / 2 3 Biological Cryo-Imaging Group MartinWalsh, Science Group Leader T he Biological Cryo-imaging Group brings together dedicated facilities for cryo-electron microscopy (cryo-EM), X-ray and super resolution light microscopies at Diamond Light Source. The electron Bioimaging Centre (eBIC) is the national centre for cryo-EM in the UK and provides a range of capabilities and supporting facilities for single particle cryo-EM, cryo-Electron Tomography (cryo-ET) and Correlative Light and Electron Microscopy (CLEM). Beamline B24 hosts a full field cryo-transmission X-raymicroscope dedicated to biological X-ray imaging and has also established cryo super-resolutionfluorescencemicroscopy, which is a joint venture betweenDiamond and theUniversity of Oxford. It provides a unique platform for correlative light and X-ray microscopy, and cryo-EM. The Biological Cryo-Imaging group continues to grow with enhancements to established instruments and new capabilities. At eBIC, there is a long list, but highlights include: 1) Roll-out of an external commissioning user program to access the correlative workflows as part of our cryo-ET access, through the use of the Leica cryo-CLEM, with first users taking advantage of this access route in 2022; 2) Access to semi-automated cryoFIB milling via the Aquilos-2 cryoFIB/SEM dual beam microscope is now fully integrated into the BAG access system. The Aquilos-2 will be further upgraded in June 2023 with the integration of fluorescence light microscopy (FLM) by means of the Meteor system from delmic. This will allow users to perform in situ CLEM experiments in the vacuum of the cryoFIB/SEM and will facilitate the identification and conformation of regions of interest before and during milling. This will greatly increase the chance that the region of interest will be retained in the milled lamellae when imaged in one of the eBIC Krios’s; 3) During 2022 the eBIC team integrated the Dectris SINGLA detector, a hybrid-pixel electron-counting camera to assess its capabilities for micro-electron diffraction experiments. The detector was installed on the Research Complex at Harwell Glacios™ 200 kV Cryo-TEM that is hosted and operated in partnership with eBIC. A coordinated effort with the DIALS group, the HeXI instrument, CCP-EM and CCP4 allowed us to establish a rapid and automated workflow for the collection of microED data. An access program is currently in development, and we expect to start with friendly users during the summer of 2023; 4) Data management and automation of data processing for users has been considerably simplified by the development of Murfey both for single particle cryo-EM and Cryo-ET. Murfey automates the data transfer and processing pipelines for both single particle cryo-EM and cryo-ET at eBIC; 5) A new on-the-fly cryo-ET processing pipeline has been implemented, which generates reconstructed 3D volumes allowing the users to assess their data as it is collected, making sure that only the best data is collected and used for further analysis; 6) eBIC is delighted to be a partner in the Horizon Europe project Fragment-Screen (https:// fragmentscreen.org/home) , with Diamond’s participation facilitated by funding from the UKRI Horizon Europe guarantee scheme. The project started on the 1 st of February 2023. B24 has also had another strong year with the beamline now fully commissioned and users able to access both instruments (B24’s full field cryo X-ray transmission microscope for soft X-ray tomography (cryoSXT) experiments and the bespoke cryo-Structured illumination Microscope (CryoSIM) for 3D super-resolution fluorescence cryo-imaging). The team are refining correlative workflows with cryoSXT and CryoEM which have been pioneered at B24. A major effort continues in fostering and growing the user community and the last year has been busy with the team raising awareness of the unique capabilities of B24 at both national and international conferences together with focused workshops to train and educate users. Simplifying the B24 user data collection and processing experience is progressing nicely with the team automating part of the workflow through further development of the B24 software pipeline. Some of the workflow and software developments have been supported through the Horizon 2020 project iNEXT-Discovery (https://inext-discovery.eu/) and successfully delivered for the wider community. The team has also developed additional functionality by providing for tomography experiments to be carried out at the iron and calcium edges. A reminder that access for European users of both eBIC and B24 continues to be supported through iNEXT-Discovery which runs till the end of July 2024. As an iNEXT-Discovery user, access and support is fully funded and European based groups are encouraged to apply through the iNEXT-Discovery user portal. Finally, the BCI user meeting continues to grow and in partnership with the CCP-EM spring symposium this year’s hybrid meeting surpassed our expectations with 225 participants in person and 484 zoom attendees attending the BCI user meeting which kicked off a very enjoyable 3 days of talks from users and the wider community. We look forward to seeing many of you at next year’s meeting which will again be hosted at the East Midlands Conference Centre in Nottingham. Amajor effort continues in fostering and growing the user community and the last year has been busywith the team raising awareness of the unique capabilities of B24 at both national and international conferences together with focusedworkshops to train and educate users. Figure 1: the B24 team. Figure 2: the eBIC team.