Diamond Annual Review 2019/20

22 23 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 1 9 / 2 0 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 1 9 / 2 0 Biological Cryo-Imaging MartinWalsh, Deputy Director of Life Sciences and Science Group Leader T he Biological Cryo-Imaging Group at Diamond brings together dedicated facilities for X-ray, light and electron microscopy. B24 is a cryo-TXM (full field cryo-transmission X-ray microscopy) beamline dedicated to biological X-ray imaging, which has established a cryo-super-resolution fluorescence microscopy facility in a joint venture between Diamond and the University of Oxford. Exploiting electrons for imaging, the electron Bio-Imaging 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. Following on fromour jam-packed first full year, the Biological Cryo-Imaging Group continues to grow and introduce new capabilities and ways of working. It has been a busy year, with both B24 and eBIC developing and expanding their capabilities. This has meant that installation, commissioning of new instrumentation and associated recruitment has remained our major focus. At eBIC during 2019, we finalised upgrades to all our Titan Krios’ with the installation and commissioning of the K3™direct electron counting detector from Gatan.TheThermoFisherAquilos TM cryofocused ionbeam(cryo-FIB)andscanning electron microscope has been fully commissioned and integrated into the eBIC user programme. As part of an extensive collaboration with Thermo Fisher and the Rosalind Franklin Institute, a cryo-capable Helios Hydra dual-beam has also been installed and commissioned. To facilitate and grow the user community, two modes of access to the cryo-FIB instruments (Scios TM and Aquilos TM ) are now in place. A rapid access route provides users with a day for cryo-FIB milling of samples, which they then take back to their home laboratory for imaging.This is followed by a standard access route, which provides three days of cryo-FIBmilling and two days of microscope time (Titan Krios) for cryo-electron tomography (cryo-ET) data collection. At B24, upgrades to the X-ray microscope in order to allow acquisition of data at higher energies and exploit phase contrast imaging at the beamline are progressing well with installation of phase contrast optics completed and currently under commissioning. The collection of dual-axis X-ray data collection leading to the reduction of missing wedge artefacts has also been successfully demonstrated and workflows continued to be improved e.g. samples can now be accepted on both conventional EM grids and autogrids. A great deal of the software focus has been towards improving correlative workflows in, for example,automationof imageregistrationbetweencryo-structured illumination microscopy (cryo-SIM) and cryo-soft X-ray tomography (cryo-SXT) through development of new algorithms. Correlative Light X-ray Microscopy (CLXM) has been enthusiastically taken up by the user community and is in high demand. In addition, we were delighted to perform our first Correlative Light and Electron Microscopy (CLEM) experiment with external users. Training for users continues to be a major activity at B24 and eBIC. Over the summer of 2019, B24 organised and delivered a Cryo-Imaging Correlative Image Data Analysis Workshop at the University of Okayama, Japan and also hosted a Super-Resolution Fluorescence Microscopy Developers Symposium at Diamond. At eBIC, hands-onworkshops continue to be the primary focus, and these remain highlypopularandheavilyoversubscribed.Thewell-establishedCryo-EMSample PreparationWorkshopcontinuestobeofferedbi-annuallyandaMicro-ED(Micro- Electron Diffraction) Workshop was held to introduce the technique to structural biologists. A series of lectures covering theoretical background and practical considerations together with hands on data collection at eBIC using EPU-D and tutorials on data reduction and analysis again created a high demand for places by participants. eBIC’s central role in training the EM community is exemplified by a recently funded extensive collaborative grant with the Astbury Centre in Leeds and other institutions. This five year grant for EM training, funded by the Wellcome Trust and MRC, will enable a significant uplift in both training courses and hands-on instrument use. Software training at eBIC continues to be carried out in collaboration with CCP-EM, although the programme has been disrupted bytheCOVID-19pandemic.However,eBICmicroscopeswere involved intherapid response to COVID-19 provided by Diamond and continued operations focused on SARS-Cov-2 research in spite of the UK lockdown. Finally, following on from the success of iNEXT (Infrastructure for NMR, Electron microscopy, and X-rays for Translational research), which was funded by the European Commission H2020 programme to provide access to structural biology infrastructure,anextendedandenhancedprogramme- iNEXT-Discovery 1 - has been funded and commenced on the 1st February 2020. The overall aim of these networks is to aid the opening and integration of existing national and regional research infrastructures to the wider European science user community. iNEXT provided state-of-the-art access to Macromolecular Crystallography (MX), Biological Solution Small Angle X-ray Scattering (BioSAXS), Nuclear Magnetic Resonance (NMR) and cryo-EM. Support for access to MX, BioSAXS and cryo- EM at Diamond has allowed us to assist users from across Europe to access our facilities and to, also, fund a number of joint research activities to further enhance and developmethods and capabilities. iNEXT-Discovery brings X-ray imaging into the fold with instrument access to both B24 at Diamond and Mistral at ALBA, Spain. B24 and Mistral are currently collaborating on developing correlative workflows for CLXM. eBIC will provide access to cryo-FIB milling, in addition to instrument access for single particle and tomography experiments, and lead the training programme for cryo-EMwithin iNEXT-Discovery. Although progress in the first quarter of 2020 has been impacted by the COVID-19 pandemic, we are continuing to work hard to further expand our bio- imaging capabilities in 2020 and look forward to growing our user community and increasing our impact in the field of 3D bio-imaging. 1. https://inext-discovery.eu/network/inext-d/home