MICROSCOPY & IMAGING
and bacteria) and their interactions with host cells, stem cell development and differentiation, autoimmune pathologies, biomaterials and their biological applications as well as anticancer drug and vaccine effects in cells. Because of the way samples are cryopreserved for CLXT, intracellular processes can be track-and-traced in 3D and at high resolution without disturbing delicate cellular structures, which allows the capture of dynamic processes such as drug delivery, pathogen clearance and cell signalling. Te power of CLXT has been recently demonstrated in a collaboration with the Kennedy Institute of Rheumatology in Oxford University[2]
where, combining
a series of imaging techniques (confocal, dSTORM and X-ray microscopy), cytotoxic T lymphocytes (CTLs) were shown to generate and transfer distinct cytotoxic multiprotein complexes, called supramolecular attack particles, to eliminate target cells. According to Professor of Immunology at the Kennedy Institute, Michael Dustin, “Beamline B24 is a very exciting resource to study exchange of information in the immune system. Matching the 25nm resolution of SXT with similar resolution of specific molecular assemblies by super resolution fluorescence such as SIM and STORM, will open up many fundamental questions in immunology and cell biology.” A number of industrial groups have also used the beamline to evaluate processes and materials. Recent work involved the evaluation and application of new vaccine formulations in mammalian cell lines to confirm antigenic potential and overall effect on host cell physiology
(manuscript in preparation). According to Dr Claire Pizzey, deputy head of the industrial liaison group at Diamond: “Te scientific insight gained using the B24 correlative microscopy approach has been of great value in the areas of vaccine development and novel therapeutics, feeding into our clients’ R&D pipelines in a timely manner. Te full-service approach, where we support our clients through experimental design, data collection, analysis and reporting, is of particular interest to our clients who recognise the benefits of the technique but many have no prior experience.”
ACCESSIBILITY FOR ACADEMIA AND INDUSTRY Continued demand to deliver correlative imaging at the cellular level in complex biological systems is currently driving further developments at the beamline. Tis will ensure high-quality data is accumulated faster and more reliably and it is anticipated that many otherwise challenging projects will benefit from its application in the future. Te microscopes at beamline B24 are accessible to both academia and industry via well-defined access routes. For academic groups, access requires the submission of a proposal: a ‘rapid access’ one for proof-of-principle studies or a ‘standard’ one for established projects; the latter enters a competitive access process that involves peer-review and award of usage shifts. Industrial groups on the other hand access the beamline via the industrial liaisons team at Diamond. It is recommended that potential users contact the B24 team to discuss requirements and plan their experiments in way that takes full advantage of what is on offer. During the Covid-19 pandemic users are still able to send their samples to B24 and conduct data collection remotely while all the necessary protocols and software instructions are fully accessible online.
Dr Ilias Kounatidis (beamline scientist at Diamond Light Source) preparing samples for cryopreservation at beamline B24
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www.scientistlive.com
ENHANCING UNDERSTANDING OF THE CELLULAR WORLD Te correlative microscopy platform available at B24 is a new tool that has been developed specifically to promote and enhance our understanding of the cellular world and its potential. Te technology on site is user-friendly and accessible and remains dynamic as it is
SIM fluorescence showing the distribution of reovirus (green) and the vesicles it occupies (red) within an infected human cell with slices of the corresponding SXT data from the same area showing the outlines of cytoplasmic vesicles and organelles (a part of the nucleus can be seen bottom right). The combination of the two datasets allowed the unambiguous identification of the vesicles involved in the trafficking of reovirus particles in the early hours post-infection
upgraded constantly in response to the latest demands of the wider biomedical community.
References [1] Kounatidis I, Stanifer ML, Phillips MA, et al. 3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway. Cell. 2020;182(2):515-530.e17.
[2] Bálint, S et al. Supramolecular attack particles are autonomous killing entities released from cytotoxic T cells. Science (New York, N.Y.) vol. 368,6493 (2020): 897-901.
Maria Harkiolaki is principal beamline scientist, B24 & Ilias Kounatidis is beamline scientist, B24, at Diamond.
www.diamond.ac.uk/B24
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