Diamond Annual Review 2021/22

30 31 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 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 Beamline B24 Combining synchrotron and laser light to 3D-image subcellular architecture under cryogenic conditions Related publication: Koronfel, M., Kounatidis, I., Mwangangi, D. M., Vyas, N., Okolo, C., Jadhav, A., Fish, T., Chotchuang, P., Schulte, A., Robinson, R. C., &Harkiolaki, M. Correlative cryo-imaging of the cellular universe with soft X-rays and laser light used to track F-actin structures inmammalian cells. Acta Crystallographica Section D Structural Biology 77 , 1479–1485 (2021).DOI: 10.1107/S2059798321010329 Publication keywords : X-ray tomography;Water-window absorption imaging; Structured illumination; Cytoskeleton; Correlative cryo-imaging W hen viewing a sample with different microscopy techniques, these often cannot be correlated because of differences in the scale and the detail of the information they capture. In the case of cells, imaging the cytoskeleton within the context of the fully hydrated cellular ultrastructure and its contents to high resolution and in 3D needs a specialised combination of microscopes that did not exist previously. Researchers wanted to understand the distribution and architecture of filamentous actin, which represents the finest and most dynamic component of the cytoskeleton. They used the Cryo Soft X-ray Tomography beamline (B24), ensuring the capture of the native ultrastructure through vitrification without the need for chemical or mechanical modification. The teamused Cryo Soft X-ray Tomography (cryoSXT) and cryo Structured Illumination Microscopy (cryoSIM) to capture the 3D ultrastructure of the cell and the chemical localisation of filamentous actin, and to unambiguously correlate these data in 3D. Their findings confirm the applicability of high-resolution cutting-edge 3D cryo-microscopy methods at beamline B24 in the study of biological processes. Theminimal disruption approach ensures the capture of physiologically relevant information that can be usedwithout concern over artefact induction due to sample processing. To date, the B24 imaging platform has been used for a number of investigations into understanding basic biological mechanisms, and also for the assessment and understanding of subjects of biomedical importance such as anti-cancer drugs and vaccine development. The subcellular universe inside living cells at physiological conditions is rich with content such as macro- and supra-molecular structures, conglomerates of vesicles and organelles alongside concentrations of inorganic and organic chemicals tethered or flowing along gradients and currents. All these constituents have a role to play (and often multiple roles) in maintaining a healthy cellular environment and responding to external stimuli as they interact with each other constantly. Because of this dynamic and multivariant subject matter, imaging one set of cellular features using one microscope invariably misses relevant features that can only be captured by different microscopy methods and the correlation of imaging data across methods becomes invaluable to fully understand context beyond localisation within an area of interest. At beamline B24, two powerful imaging methods have been paired and used here to track and understand the role of filamentous actin in human fibroblasts 1 (Fig. 1). Cryo-Soft X-ray Tomography (cryoSXT) is a relatively young technique that has developed following progress in cryogenic sample preparation in the previous two decades with a view of preparing cellular material that retains its native ultrastructure in a form that can withstand the radiation exposure needed to capture high content high resolution 3D imaging. CryoSXT uses X-rays in the ‘water window’ of the spectrum where carbon-rich biological material such as membranes and vesicles absorb X-rays readily in contrast to the oxygen-rich medium that surrounds them leaving as a result an imprint of their conformation on the detector that receives the transmitted rays 2 (Fig. 2a). The principle verymuch reflects the application of CT in amedical setting but in this case the microscope is adapted to capture the intracellular environment. The method has proven to be an invaluable tool in assessing cellular response but lacks information on chemical localisation, namely it cannot provide the distribution of chemicals or proteins of interest. This has been achieved at B24 beamline through the combined use of fluorescence microscopy and specifically cryo-Structured Illumination Microscopy (cryoSIM) which allows the capture of chemical localisation beyond the diffraction limit through fluorescence excited via laser light. The two microscopes work synergistically and multiply the information content of the data acquired by providing direct and unambiguous 3D correlation 3 (Fig. 2b & 2c). When studying the cytoskeleton, it is often challenging to capture the finer and most dynamic most dynamic element of its components (filamentous actin) in 3D and to nanometer resolution. Although cryoSXT is an excellent tool for studying subcellular architecture, filamentous actin is difficult to decipher in an X-ray tomogram because of its low contrast profile precisely because of the finer and transient structures it creates. Using the B24 integrated imaging pipeline the authors were able to capture the chemical information needed within the cellular ultrastructure and to unambiguously localise filamentous actin structures such as supra-filaments, networks and vesicle cradles. In doing so they have established the correlative method as a valuable tool in this type of research and have enabled the gathering of information beyond what each method would be able to provide separately. From a biological standpoint, they expected filamentous actin to play an integral role in cellular functions such as vesicle budding and transport, cell-to-cell contact, organelle network and substructure support as well as nuclear localisation and gene regulation 4 . Using correlated imaging they observed filamentous actin deployed in the fringes of the cell cytoplasm where contact with neighbours required the traditional actin networks that give rise to filopodia (Fig. 2c). However, its role is clearly more diverse as they examined the perinuclear area where they were able to clearly document filamentous actin-dependent vesicle transport (Fig. 3) through extensive networks of filamentous actin that connect and support organelles such as mitochondria and endosomes. It was also present within multivesicular bodies, but further work will need to be done to fully characterise this specific actin tropism, be it an incident of biological recycling or an active form of remodelling for purposes yet undisclosed. The work presented here demonstrated the importance of technology development in the field of correlative biological cryo-imaging and has provided a proven imaging platform for the study of the cytoskeleton inside fully hydrated cells at near-physiological states. References: 1. Okolo, C. A. A guide into the world of high-resolution 3D imaging: the case of soft X-ray tomography for the life sciences. Biochemical Society Transactions 50 , 649–663 (2022). DOI: 10.1042/BST20210886 2. Harkiolaki, M. et al. Cryo-soft X-ray tomography: using soft X-rays to explore the ultrastructure of whole cells. Emerging Topics in Life Sciences 2 , 81–92 (2018). DOI: 10.1042/ETLS20170086 3. Kounatidis, I. et al. 3D Correlative cryo-structured illumination fluorescence and soft X-ray microscopy elucidates reovirus intracellular release pathway. Cell 182 , 515-530.e17 (2020). DOI: 10.1016/j. cell.2020.05.051 4. Dominguez, R. et al. Actin structure and function. Annual Review of Biophysics 40 , 169–186 (2011). DOI: 10.1146/annurev- biophys-042910-155359 Corresponding author : Maria Harkiolaki, Diamond Light Source, maria.harkiolaki@diamond.ac.uk Figure 1: (a) 2D soft X-ray mosaic of vitrified human fibroblasts (two cells seen here on a perforated carbon support film; the nuclei appear as round areas of low signal with defined nucleoli while the cytoplasm shows more content such as organelles and substructures; (b) the same mosaic with filamentous actin distribution as captured with cryoSIM and clearly defining the cytoskeletal architecture that is missing from the naked SXT data. Data was collected at beamline B24. Scale bar is 10 μm. Figure 2: (a) A 2D slice from a 3D tomogram showing the perinuclear area inside a human fibroblast; Organelles visible include mitochondria, endosomes, multivesicular bodies lipid droplets and associated endoplasmic reticulum; (b) & (c); cytoplasmic area within the same cell population showing the information content of the naked SXT data (b) and the one enriched with SIM data (c); where a vesicle is clearly seen engulphed by filamentous actin (top right) and the cell-to-cell contact of neighbouring cells delineated as an edge-defining line on the upper left side of both panels. Figure 3: (a) SIM data showing actin organisation; (b) the corresponding area captured through SXT and (c) the overlay of segmented fluorescence actin signal in the context of the SXT data.