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The first year of the Biological Cryo-Imaging Group at Diamond has been a busy one, with both B24 and eBIC developing and expanding their capabilities.
![Figure 1: Micro Electron diffraction at eBIC. Left: Representative Images from the Scios™
<br/>cryo-FIB scanning electron microscope: (a) lysozyme crystal before milling and after (b, c).
<br/>Transmission EM image of the milled Lamella (d). [Scale bars: a and b, 4 mm, c, 30 mm, d, 3
<br/>mm]. Right: An electron diffraction image from the lysozyme lamella. The edges of the image
<br/>correspond to ~ 1.8 Å resolution.](/dam/jcr:3ba8474b-acfc-4005-a2cd-b6e24b4eb485/p22img1.jpg)
The resolution revolution in cryo-EM has made a massive impact on the world of structural biology and this has, in no small part, been driven by detector developments, in particular direct electron detectors for which the first commercial products were made available in 2012. Improvements in detector frame rates, detection quantum efficiency and active area are being pursued by a number of groups both in academia and industry. At the end of 2018, eBIC upgraded two of its Titan Krios microscopes with the K3™ direct electron detector from Gatan, which has been keenly anticipated by the community at large. The K3™ detector has a 1.6 times bigger active area than its predecessor and can operate at 1,500 frames/second, an increase in throughput of 3.75 over the previous fastest detector on the market (the K2™, also from Gatan). The capabilities offered by these upgrades are already making a significant impact, as the increased frame rates allow shorter counting acquisition times leading to a two-fold increase in the number of movies collected per hour. This, in combination with the increased detector size of the K3, means that substantially more data can be collected in the same time than with the previous generation of detectors, maximising microscope output. The implementation of new data collection strategies, using the microscope image shift coils instead of stage movements to navigate between acquisition areas, have increased throughput further.
Interest from industry in cryo-EM has also grown at a rapid rate. Access has been facilitated by the Industrial Liaison Office at Diamond, who have a strong track record in providing industry access to Diamond beamlines, in particular the suite of Macromolecular Crystallography (MX) beamlines. Thus, our first industry users followed shortly after the academic programme started in June 2015. The strong demand from industry led to the establishment in 2018 of an exciting new partnership between Diamond and Thermo Fisher Scientific to serve the needs of industry in parallel to our academic programme. This has enabled us to provide two new dedicated microscopes, a Thermo Fisher Scientific Krios G3i cryo-TEM and a 200 kV Glacios cryo-TEM. The industrial team at eBIC, together with embedded Thermo Fisher staff, will provide support and guidance to industrial researchers in sample preparation, cryo-EM sample screening, and high-end data collection. First users of the industry Titan Krios were welcomed in November 2018.
A full user programme was delivered from April 2018 at B24 and the interest from user groups continues to grow steadily (the beamline was heavily oversubscribed in the last call for proposals). The X-ray microscope has benefitted from the incorporation and full commissioning of a 25 nm zone plate which is currently available to users. The integration of a bespoke cryo-super resolution fluorescence module at B24 (an international first!) is also in high demand and has been developed to offer both cryo-Structured Illumination Microscopy (cryo-SIM) and dSTORM. This allows a unique correlative imaging workflow, whereby B24 users first collect 3D fluorescence imaging data at optical resolutions beyond the diffraction limit. This allows areas of biological and chemical interest to be identified before the same sample is taken to the cryo-transmission X-ray microscope where 3D X-ray data can be taken on the same areas of interest and directly correlated with the existing 3D fluorescent microscopy data. The B24 team are working with the data analysis team at Diamond to improve tomographic reconstruction results, and with the developers of easy cell-correlative light to electron microscopy (eC-CLEM) an open source software for Correlative Light Electron Microscopy (CLEM) to improve correlative microscopy workflows to improve correlative workflows, which has led to a collaborative PhD project due to start in September 2019. Currently all fiducialised X-ray data is automatically processed into tomograms allowing users to interactively evaluate samples and refine data collection strategies during their visit. All fluorescence data is also processed on site during data collection, and the focus is now on automation of data correlation across modalities.
Training for users continues to be a major activity at B24 and eBIC. At B24, a focus has been on hands-on sessions covering sample preparation, data collection, data processing and imaging data correlation. Software training at eBIC has been carried out in collaboration with CCP-EM, which has meant a busy schedule of basic and specialised courses in cryo-EM software such as Relion 3. The third instalment of eBIC’s ‘hands-on’ training workshops in sample preparation for cryo-EM was again heavily oversubscribed and well received. This was complemented by focused training of eBIC users and Block Allocation Groups (BAGs).
Finally, looking to the future and the potential benefits and opportunities that will arise from the Diamond-II machine upgrade for imaging, were avidly discussed at a joint workshop between the BioImaging and Imaging and Microscopy groups at Diamond. The workshop’s aims were to introduce the scope of the Diamond machine upgrade, bring together experts in the field and Diamond users to identify key science areas that would benefit from the upgrade as a contribution to the development of the science case. The workshop provided a timely forum for experts and Diamond users to discuss the potential for upgrading existing instruments and the opportunities for new beamlines. Of particular interest for the BioImaging community were discussions centred around the potential science drivers for a dedicated cryo-imaging beamline for biology, which would exploit cryo-ptychography, holography and fluorescence microscopy.
In summary, the BioImaging group at Diamond continued to grow in 2018, as has the impact on biology of the user programme. We look forward to further expanding both our BioImaging capabilities and their impact in 2019.
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