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Instruments / Technical Area	Publication Details	Published
B24-Cryo Soft X-ray Tomography	A combination of soft X-ray and laser light sources offer 3D high content information on the native state of the cellular environment Chidinma A. Okolo , Thomas M. Fish , Kamal L. Nahas , Archana C. Jadhav , Nina Vyas , Adam Taylor , Maria Harkiolaki Journal Of Physics: Conference Series 2380 14th International Conference on Synchrotron Radiation Instrumentation (SRI 2021) DOI: 10.1088/1742-6596/2380/1/012042 Open Access Show Abstract ▼ Abstract: Beamline B24 is a life sciences correlative cryo-imaging beamline at Diamond Light Source. B24 uses a combination of conventional and super-resolution visible-light fluorescence microscopy and soft X-ray tomography (cryoSXT) to provide 3D imaging of the cellular landscape at a resolution up to 25 nm in cryo-preserved biological samples up to 12 μm thick. B24 offers user-friendly, semi-automated 3D correlative cryo-imaging through an integrated platform of methods that encompass (a) sample preparation and evaluation, (b) data collection and processing and (c) data analysis and correlation. CryoSXT fills the current resolution gap between fluorescence and electron microscopy while cryo-structured illumination microscopy provides the additional dimension of chemical localization within the same cellular ultrastructure captured by cryoSXT. Beamline instruments can be accessed biannually by academics and industry globally through peer-reviewed standard and rapid access proposal processes. The B24 user base is primarily academic research groups studying cell function and cytopathology in biological systems ranging from viruses and algae to mammalian cells and proto-tissue complexes. Future work will consolidate development efforts and experiences gained thus far to enable high-throughput data collection. Special emphasis is placed on the delivery of other integrated advanced imaging methods such as X-ray absorption near-edge spectroscopy and phase contrast.	Dec 2022
B24-Cryo Soft X-ray Tomography	Synchrotron radiation and laser light microscopy partnership for the study of biological systems: the case of Soft X-ray Tomography and structured illumination microscopy at cryogenic temperatures Maria Harkiolaki , Nina Vyas , Claire Pizzey , Thomas Fish , Archana Jadhav , Kamal Nahas , Chidinma Okolo Microscopy And Microanalysis 28, 1328 - 1330 Microscopy & Microanalysis 2022: B04 - Correlative and Multimodal Microscopy and Analysis DOI: 10.1017/S1431927622005463	Aug 2022
B24-Cryo Soft X-ray Tomography	Near-native state imaging by cryo-soft-X-ray tomography reveals remodelling of multiple cellular organelles during HSV-1 infection Kamal Nahas , Viv Connor , Katharina M. Scherer , Clemens F. Kaminski , Maria Harkiolaki , Colin M. Crump , Stephen C. Graham Plos Pathogens 18 DOI: 10.1371/journal.ppat.1010629 Diamond Proposal Number(s): [18925, 19958, 21485, 23508] Open Access Show Abstract ▼ Abstract: Herpes simplex virus-1 (HSV-1) is a large, enveloped DNA virus and its assembly in the cell is a complex multi-step process during which viral particles interact with numerous cellular compartments such as the nucleus and organelles of the secretory pathway. Transmission electron microscopy and fluorescence microscopy are commonly used to study HSV-1 infection. However, 2D imaging limits our understanding of the 3D geometric changes to cellular compartments that accompany infection and sample processing can introduce morphological artefacts that complicate interpretation. In this study, we used soft X-ray tomography to observe differences in whole-cell architecture between HSV-1 infected and uninfected cells. To protect the near-native structure of cellular compartments we used a non-disruptive sample preparation technique involving rapid cryopreservation, and a fluorescent reporter virus was used to facilitate correlation of structural changes with the stage of infection in individual cells. We observed viral capsids and assembly intermediates interacting with nuclear and cytoplasmic membranes. Additionally, we observed differences in the morphology of specific organelles between uninfected and infected cells. The local concentration of cytoplasmic vesicles at the juxtanuclear compartment increased and their mean width decreased as infection proceeded, and lipid droplets transiently increased in size. Furthermore, mitochondria in infected cells were elongated and highly branched, suggesting that HSV-1 infection alters the dynamics of mitochondrial fission/fusion. Our results demonstrate that high-resolution 3D images of cellular compartments can be captured in a near-native state using soft X-ray tomography and have revealed that infection causes striking changes to the morphology of intracellular organelles.	Jul 2022
B24-Cryo Soft X-ray Tomography	Contour : A semi-automated segmentation and quantitation tool for cryo-soft-X-ray tomography Kamal Nahas , João Ferreira Fernandes , Nina Vyas , Colin Crump , Stephen Graham , Maria Harkiolaki Biological Imaging 2 DOI: 10.1017/S2633903X22000046 Diamond Proposal Number(s): [18925, 19958, 21485, 23508] Open Access Show Abstract ▼ Abstract: Cryo-soft-X-ray tomography is being increasingly used in biological research to study the morphology of cellular compartments and how they change in response to different stimuli, such as viral infections. Segmentation of these compartments is limited by time-consuming manual tools or machine learning algorithms that require extensive time and effort to train. Here we describe Contour, a new, easy-to-use, highly automated segmentation tool that enables accelerated segmentation of tomograms to delineate distinct cellular compartments. Using Contour, cellular structures can be segmented based on their projection intensity and geometrical width by applying a threshold range to the image and excluding noise smaller in width than the cellular compartments of interest. This method is less laborious and less prone to errors from human judgement than current tools that require features to be manually traced, and it does not require training datasets as would machine-learning driven segmentation. We show that high-contrast compartments such as mitochondria, lipid droplets, and features at the cell surface can be easily segmented with this technique in the context of investigating herpes simplex virus 1 infection. Contour can extract geometric measurements from 3D segmented volumes, providing a new method to quantitate cryo-soft-X-ray tomography data. Contour can be freely downloaded at github.com/kamallouisnahas/Contour.	May 2022
B24-Cryo Soft X-ray Tomography	Epitope length variants balance protective immune responses and viral escape in HIV-1 infection Phillip Pymm , Stefan Tenzer , Edmund Wee , Mirjana Weimershaus , Anne Burgevin , Simon Kollnberger , Jan Gerstoft , Tracy M. Josephs , Kristin Ladell , James E. Mclaren , Victor Appay , David A. Price , Lars Fugger , John I. Bell , Hansjörg Schild , Peter Van Endert , Maria Harkiolaki , Astrid K. N. Iversen Cell Reports 38 DOI: 10.1016/j.celrep.2022.110449 Open Access Show Abstract ▼ Abstract: Cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses to a single optimal 10-mer epitope (KK10) in the human immunodeficiency virus type-1 (HIV-1) protein p24Gag are associated with enhanced immune control in patients expressing human leukocyte antigen (HLA)-B∗27:05. We find that proteasomal activity generates multiple length variants of KK10 (4–14 amino acids), which bind TAP and HLA-B∗27:05. However, only epitope forms ≥8 amino acids evoke peptide length-specific and cross-reactive CTL responses. Structural analyses reveal that all epitope forms bind HLA-B∗27:05 via a conserved N-terminal motif, and competition experiments show that the truncated epitope forms outcompete immunogenic epitope forms for binding to HLA-B∗27:05. Common viral escape mutations abolish (L136M) or impair (R132K) production of KK10 and longer epitope forms. Peptide length influences how well the inhibitory NK cell receptor KIR3DL1 binds HLA-B∗27:05 peptide complexes and how intraepitope mutations affect this interaction. These results identify a viral escape mechanism from CTL and NK responses based on differential antigen processing and peptide competition.	Mar 2022
B24-Cryo Soft X-ray Tomography	Identification of distinct cytotoxic granules as the origin of supramolecular attack particles in T lymphocytes Hsin-Fang Chang , Claudia Schirra , Momchil Ninov , Ulrike Hahn , Keerthana Ravichandran , Elmar Krause , Ute Becherer , Stefan Balint , Maria Harkiolaki , Henning Urlaub , Salvatore Valitutti , Cosima T. Baldari , Michael L. Dustin , Reinhard Jahn , Jens Rettig Nature Communications 13 DOI: 10.1038/s41467-022-28596-y Open Access Show Abstract ▼ Abstract: Cytotoxic T lymphocytes (CTL) kill malignant and infected cells through the directed release of cytotoxic proteins into the immunological synapse (IS). The cytotoxic protein granzyme B (GzmB) is released in its soluble form or in supramolecular attack particles (SMAP). We utilize synaptobrevin2-mRFP knock-in mice to isolate fusogenic cytotoxic granules in an unbiased manner and visualize them alone or in degranulating CTLs. We identified two classes of fusion-competent granules, single core granules (SCG) and multi core granules (MCG), with different diameter, morphology and protein composition. Functional analyses demonstrate that both classes of granules fuse with the plasma membrane at the IS. SCG fusion releases soluble GzmB. MCGs can be labelled with the SMAP marker thrombospondin-1 and their fusion releases intact SMAPs. We propose that CTLs use SCG fusion to fill the synaptic cleft with active cytotoxic proteins instantly and parallel MCG fusion to deliver latent SMAPs for delayed killing of refractory targets.	Feb 2022
B24-Cryo Soft X-ray Tomography	Correlative cryo-imaging of the cellular universe with soft X-rays and laser light used to track F-actin structures in mammalian cells Mohamed A. Koronfel , Ilias Kounatidis , Dennis M. Mwangangi , Nina Vyas , Chidinma Okolo , Archana Jadhav , Tom Fish , Phatcharin Chotchuang , Albert Schulte , Robert Robinson , Maria Harkiolaki Acta Crystallographica Section D Structural Biology 77, 1479 - 1485 DOI: 10.1107/S2059798321010329 Diamond Proposal Number(s): [23033, 23073] Open Access Show Abstract ▼ Abstract: Imaging of actin filaments is crucial due to the integral role that they play in many cellular functions such as intracellular transport, membrane remodelling and cell motility. Visualizing actin filaments has so far relied on fluorescence microscopy and electron microscopy/tomography. The former lacks the capacity to capture the overall local ultrastructure, while the latter requires rigorous sample preparation that can lead to potential artefacts, and only delivers relatively small volumes of imaging data at the thinnest areas of a cell. In this work, a correlative approach utilizing in situ super-resolution fluorescence imaging and cryo X-ray tomography was used to image bundles of actin filaments deep inside cells under near-native conditions. In this case, fluorescence 3D imaging localized the actin bundles within the intracellular space, while X-ray tomograms of the same areas provided detailed views of the local ultrastructure. Using this new approach, actin trails connecting vesicles in the perinuclear area and hotspots of actin presence within and around multivesicular bodies were observed. The characteristic prevalence of filamentous actin in cytoplasmic extensions was also documented.	Dec 2021
B24-Cryo Soft X-ray Tomography I14-Hard X-ray Nanoprobe	Single-cell chemistry of photoactivatable platinum anticancer complexes Elizabeth M. Bolitho , Carlos Sanchez-Cano , Huayun Shi , Paul D. Quinn , Maria Harkiolaki , Cinzia Imberti , Peter J. Sadler Journal Of The American Chemical Society 8 DOI: 10.1021/jacs.1c08630 Diamond Proposal Number(s): [22696, 21505, 25824] Open Access Show Abstract ▼ Abstract: The Pt(IV) prodrug trans, trans, trans-[Pt(pyridine)2(N3)2(OH)2] (Pt1) and its coumarin derivative trans, trans, trans-[Pt(pyridine)2(N3)2(OH)(coumarin-3-carboxylate)] (Pt2) are promising agents for photoactivated chemotherapy. These complexes are inert in the dark but release Pt(II) species and radicals upon visible light irradiation, resulting in photocytotoxicity toward cancer cells. Here, we have used synchrotron techniques to investigate the in-cell behavior of these prodrugs and visualize, for the first time, changes in cellular morphology and Pt localization upon treatment with and without light irradiation. We show that photoactivation of Pt2 induces remarkable cellular damage with extreme alterations to multiple cellular components, including formation of vacuoles, while also significantly increasing the cellular accumulation of Pt species compared to dark conditions. X-ray absorption near-edge structure (XANES) measurements in cells treated with Pt2 indicate only partial reduction of the prodrug upon irradiation, highlighting that phototoxicity in cancer cells may involve not only Pt(II) photoproducts but also photoexcited Pt(IV) species.	Nov 2021
B24-Cryo Soft X-ray Tomography	Correlative 3D cryo X-ray imaging reveals intracellular location and effect of designed antifibrotic protein-nanomaterial hybrids Johannes Groen , Ana R. Palanca , Antonio Aires , Javi Conesa , David Maestro , Stefan Rehbein , Maria Harkiolaki , Ana Villar , Aitziber L. Cortajarena , Eva Pereiro Chemical Science DOI: 10.1039/D1SC04183E Diamond Proposal Number(s): [25162] Open Access Show Abstract ▼ Abstract: Revealing the intracellular location of novel therapeutic agents is paramount for the understanding of their effect at the cell ultrastructure level. Here, we apply a novel correlative cryo 3D imaging approach to determine the intracellular fate of a designed protein-nanomaterial hybrid with antifibrotic properties that shows great promise in mitigating myocardial fibrosis. Cryo 3D structured illumination microscopy (cryo-3D-SIM) pinpoints the location and cryo soft X-ray tomography (cryo-SXT) reveals the ultrastructural environment and subcellular localization of this nanomaterial with spatial correlation accuracy down to 70 nm in whole cells. This novel high resolution 3D cryo correlative approach unambiguously locates the nanomaterial after overnight treatment within multivesicular bodies which have been associated with endosomal trafficking events by confocal microscopy. Moreover, this approach allows assessing the cellular response towards the treatment by evaluating the morphological changes induced. This is especially relevant for the future usage of nanoformulations in clinical practices. This correlative super-resolution and X-ray imaging strategy joins high specificity, by the use of fluorescence, with high spatial resolution at 30 nm (half pitch) provided by cryo-SXT in whole cells, without the need of staining or fixation, and can be of particular benefit to locate specific molecules in the native cellular environment in bio-nanomedicine.	Oct 2021
B24-Cryo Soft X-ray Tomography	Correlative imaging using super‐resolution fluorescence microscopy and soft X‐ray tomography at cryogenic temperatures provides a new way to assess virosome solutions for vaccine development Chidinma A. Okolo , Archana Jadhav , Patrick Phillips , Maud Dumoux , Amanda A. Mcmurray , Vishwas D. Joshi , Claire Pizzey , Maria Harkiolaki Journal Of Microscopy DOI: 10.1111/jmi.13054 Diamond Proposal Number(s): [24622] Open Access Show Abstract ▼ Abstract: Active Virosomes (AVs) are derivatives of viruses, broadly similar to ‘parent’ pathogens, with an outer envelope that contains a bespoke genome coding for 4–5 viral proteins capable of eliciting an antigenic response. AVs are essentially novel vaccine formulations that present on their surface selected viral proteins as antigens. Once administered, they elicit an initial “anti-viral” immune response. AVs are also internalised by host cells where their cargo viral genes are used to express viral antigen(s) intracellularly. These can then be transported to the host cell surface resulting in a second wave of antigen exposure and a more potent immuno-stimulation. A new 3D correlative microscopy approach is used here to provide a robust analytical method for characterisation of Zika and Chikungunya-derivatised AV populations including vesicle size distribution and variations in antigen loading. Manufactured batches were compared to assess the extent and nature of batch-to-batch variations. We also show preliminary results that verify antigen expression on the surface of host cells. We present here a reliable and efficient high-resolution 3D imaging regime that allows the evaluation of the microstructure and biochemistry of novel vaccine formulations such as AVs.	Aug 2021

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