32 33 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 0 / 2 1 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 0 / 2 1 Figure 1: (a) Image of the cryo-SXT microscope and (b) the cryo-SIMmodalities on site at Diamond B24. Figure panels reproduced from Kounatidis et al. (2020). Biological Cryo-Imaging Group Beamline B24 Anovel correlative 3Dmicroscopy platformfor studying cells in their native state Related publication: Kounatidis I., Stanifer M. L., Phillips M. A., Paul-Gilloteaux P., Heiligenstein X.,Wang H., Okolo C. A., FishT. M., SpinkM. C., Stuart D. I., Davis I., Boulant S., Grimes J. M., Dobbie I. M. &Harkiolaki M. 3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway. Cell 182 , 515-530.e17 (2020). DOI: https://doi.org/10.1016/j.cell.2020.05.051 Publication keywords: Correlative imaging; Structured illuminationmicroscopy; X-ray tomography; Reovirus biology; Correlative light/X-ray tomography (CLXT) R esearchers havedevelopedanewtechnique for studying cells in their native state.Thegoalwas toobtainhigh-quality imagingdata from cellswithouttheneedforsectioningorchemicalfixation.Thenewmethodavoidsanytreatmentthatwoulddisturbcellstructure,sothat no artefacts (errors) are introduced into the images. To demonstrate this novel correlative microscopy platform’s effectiveness, the team studied the early stages of cell infection by reoviruses. Although the specific viruses have been studied extensively, there is a debate regarding the method of infection. This research focused on the way that the virus escapes from vesicles, a required step for replication. At beamline B24, using a correlative imaging approach by combining soft X-ray tomographywith super resolutionmicroscopy in cryogenic conditions, the teamtracked the infectionmechanism.The results revealed that the virus had already escaped from the host vesicles two hours after infection, with the vesicles preserving their circular shape, suggesting a gentle, pore-based exitmechanismfor the virus. Reoviruses are valuable tools that could be engineered to express proteins and have the potential to be used in vaccines. Knowing the infection mechanismwill facilitate their handling and manipulation for biomedical purposes. The new imaging platform has also been used to validate anticancercompounds,studycellstructureduringdevelopmentandinvestigateclearanceofhumanpathogenicmicroorganismbyimmunehost cells.Thework is theoutcomeof a collectiveeffort betweenDiamond Light Sourceand researchgroups and facilities across Europe, including the University of Oxford, Heidelberg University Hospital, the Université de Nantes and CryoCapCell. Imaging is currently going through an evolutionary process with many new imaging techniques providing more details with greater sensitivity, thereby elucidating critical biomedical topics. Currently, electron microscopy (EM) techniques provide details of cellular ultrastructure at a resolution level of typically 2-5 nmby using scanning or transmission EM.This valuable information is coming though with a cost, the limited technical penetration depth, which introduces the necessity of sample sectioning accompanied by chemical fixation steps (with the possibility of disturbing the cellular ultrastructure and the introduction of fixation artefacts) or laborious cryogenic workflows. At B24, a new alternative imaging platform has been developed in order to obtain high-quality 3D imaging data from cells preserved in their native state, avoiding the introduction of artefacts but also allowing the cross-correlation of data coming from complimentary imaging techniques. The core of the new correlative platform is the combination of a cryo-soft X-ray tomography (cryo-SXT) microscope with a bespoke cryo-structured illumination microscope (cryo-SIM) (Fig. 1). The cryo-SXT microscope allows imaging of 3D cellular structures of up to 10 μm in thickness at a spatial resolution down to 25 nm using radiation deriving from a bending magnet at the synchrotron 1 . The purpose built 3D-cryoSIM complements the tomographic data by providing 3D super resolution fluorescence data on chemical localisation based on the fluorescence of cellular trackers used. The reconstructed data provide high contrast, low-background imaging details even below 200 nm surpassing the limitations of conventional light microscopy 2 . The combination of the two imaging techniques, followed by a cross-correlation of the provided data, delivers a comprehensive view of both cellular ultrastructure andmolecular organisation over extended cellular volumes. The new imaging platform has been used to elucidate the infection route of reovirus; an advanced experimental model for viral pathogenesis with potential applications in vaccines development 3 and anticancer treatments 4 . Despite reoviruses being extensively studied, certain steps of the virus entry mode and delivery of reovirus particles inside the cell remain poorly understood. At B24 the previously unsolved questions related to the early stages of reovirus infectionwere clarified including details of: i) how soon after infection viral particles get released in the cell, ii) where inside the cells those particles are released and iii) are the organelle-carriers of the viruses morphologically affected by the trafficking and the subsequent release of the viral particles? In the present study human cells were grown on gold grids and subsequently infected with reovirus following cryo-preservation of the samples by plunge freezingatspecifictimepoints(1,2and4hoursupon infection).Thesampleswere then scanned using both cryo-SIM and cryo-SXT microscopes at B24 to provide a comprehensive structural and functional, view of the intracellular organisation. This approach facilitated the determination of the location of both the virus and equally important of its carrier vesicles at each of the above time points. Virus detection was enabled with the use of fluor-labelled reoviruses strains while the use of a specific chemical reporter allowed tracking and evaluation of membrane- compromised and/or partially quenched carrier organelles. At 1 hour post-infection (PI) the 3D imaging data from the cryo-SIM revealed the presence of intracellular vesicles positive for reovirus, and those structures are recorded to increase further both in number and size by 2 hours PI, gradually forming large vesicles located perinuclearly at 4 hours (Fig. 2). The accumulation of the red fluor membrane disturbance reporter denotes that by 1 hour PI reovirus started to disrupt the membranes of the carrier organelles. Whether the above infection process affects the general morphology of the carrier vesicles and/or the overall landscape of the cell was still a question. Structural 3D data at higher resolution could elucidate any possible intracellular structural effects and these wereavailablebytheuseofthecryo-SXTmicroscope.Therefore,thesamesamples were imaged with X-rays allowing us to define the structure of cellular organelles across extended perinuclear areas of the cytoplasm, not accessible with other nanometer-resolution techniques. The following step is the correlation of the of cryo-SIM and the cryo-SXT data leading to a complete view of both carriers and virus alongside the surrounding cellular micro-environment using specific registration imaging software namely eC-CLEM 5 . The combination of the two data sets revealed the presence of simple vesicles carrying viral load at all the time points were checked, allowing viral particles to spread from the vesicle to the cytoplasmwith a subpopulation of them gradually enlarging and forming multi-vesicular bodies (MVBs) trackable from 2 hours after infection (Fig. 3). Another subpopulation of carrier vesicles showed distinctive, carbon-rich domed structures available only in infected samples from 2 hours after infection. Interestingly, the outer membrane of carriers vesicle remain relatively consistent suggesting that virus escapes without disrupting the membrane (Fig. 3). A possible route for the escape of virus is through a pore-based machinery that selectively allows the exit of the virus without compromising the vesicles membrane. Beyond the reovirus experimental evidence, the imaging technology at B24 so far has been used to study a wide range of biomedical topics ranging from host- pathogen interaction topics (viruses, fungi or bacteria affecting immune cells) and autoimmune diseases, to stem cell development and differentiation as well as the effect of vaccines on cell morphology. The common request for all of the above studies was the requirement for imaging at the appropriate resolution combined with the preservation of the cellular ultrastructure, which the newly developed imaging technology at B24 can now cater for. The imagingplatformwillcontinuetobe improvedaimingforahigherdegree of automation in the near future and in this respect, the experimental demands and the feedback of the user community have proved valuable. Importantly, the method can be used as a scaffold that can combine other advanced imaging techniques like electron microscopy, providing further opportunities for the development of multi-modal imaging technologies and serving the demands of the wider biomedical field. References: 1. Harkiolaki M. et al. Cryo-soft X-ray tomography: using soft X-rays to explore the ultrastructure of whole cells. Emerg. Top. Life Sci. 2 , 81–92 (2018). DOI: 10.1042/ETLS20170086 2. Schermelleh L. et al. SubdiffractionMulticolor Imaging of the Nuclear Periphery with 3D Structured IlluminationMicroscopy. Science (80-. ). 320 , 1332–1336 (2008). DOI: 10.1126/science.1156947 3. Demidenko A. A. et al. Engineering recombinant reoviruses with tandem repeats and a tetravirus 2A-like element for exogenous polypeptide expression. Proc. Natl. Acad. Sci. 110 , E1867--E1876 (2013). DOI: 10.1073/pnas.1220107110 4. Thirukkumaran C. et al. OncolyticViralTherapy Using Reovirus BT - Gene Therapy of Solid Cancers: Methods and Protocols. in (eds.WaltherW. et al.) 187–223 (Springer NewYork, 2015). DOI: 10.1007/978-1-4939-2727-2_12 5. Paul-Gilloteaux P. et al. eC-CLEM: flexiblemultidimensional registration software for correlativemicroscopies. Nat. Methods 14 , 102–103 (2017). DOI: 10.1038/nmeth.4170 Funding acknowledgement: This work was carried out with the support of Diamond Light Source, instrument B24 (proposals MX21046 andMX18314). Corresponding author: Dr Ilias Kounatidis, Diamond Light Source,
[email protected] Figure 2: Cryo-SIM images of green-labelled virus in human cells expressing membrane disturbance marker in distinct vesicles in red (a) at non-infected cell, (b) at 1 hour (c) at 2 hours (d) at 4 hours after infection. Figure panels reproduced from Kounatidis et al. (2020). Figure 3: (a) X-ray data from a cellular region 2hr after infection. Yellow arrow indicates virus-induced carbon-rich substructures and blue arrows denote vesicle areas within MVBs that remain impermeable. The same region with correlated SIM data (b) red for the membrane marker (c) green for the viral presence (d) with both fluorescent signals overlaid. Figure panels reproduced from Kounatidis et al. (2020).