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Correlative cryo-structured illumination fluorescence microscopy and soft X-ray tomography elucidates reovirus intracellular release pathway

View ORCID ProfileIlias Kounatidis, View ORCID ProfileMegan L Stanifer, Michael A. Phillips, View ORCID ProfilePerrine Paul-Gilloteaux, View ORCID ProfileXavier Helligenstein, View ORCID ProfileHongchang Wang, View ORCID ProfileChidinma A. Okolo, View ORCID ProfileThomas M. Fish, Matthew C. Spink, View ORCID ProfileDavid I. Stuart, View ORCID ProfileIlan Davis, View ORCID ProfileSteeve Boulant, View ORCID ProfileJonathan M. Grimes, View ORCID ProfileIan M. Dobbie, View ORCID ProfileMaria Harkiolaki
doi: https://doi.org/10.1101/2020.01.13.904623
Ilias Kounatidis
1Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK
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Megan L Stanifer
2Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany
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Michael A. Phillips
3Micron Advanced Imaging Consortium, Department of Biochemistry, University of Oxford, South Parks Rd, Oxford OX1 3QU, UK
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Perrine Paul-Gilloteaux
4SFR-Santé, INSERM, CNRS, UNIV Nantes, CHU Nantes, Nantes, France
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Xavier Helligenstein
5CryoCapCell, 155 bd de l’hôpital, 75013 Paris, France
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Hongchang Wang
1Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK
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Chidinma A. Okolo
1Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK
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  • ORCID record for Chidinma A. Okolo
Thomas M. Fish
1Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK
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Matthew C. Spink
1Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK
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David I. Stuart
1Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK
6Division of Structural Biology, The Henry Wellcome Building for Genomic Medicine, Roosevelt Drive, Oxford, OX3 7BN, UK
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Ilan Davis
3Micron Advanced Imaging Consortium, Department of Biochemistry, University of Oxford, South Parks Rd, Oxford OX1 3QU, UK
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Steeve Boulant
7Department of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany
8Research Group “Cellular polarity and viral infection”, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
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Jonathan M. Grimes
1Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK
6Division of Structural Biology, The Henry Wellcome Building for Genomic Medicine, Roosevelt Drive, Oxford, OX3 7BN, UK
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Ian M. Dobbie
3Micron Advanced Imaging Consortium, Department of Biochemistry, University of Oxford, South Parks Rd, Oxford OX1 3QU, UK
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Maria Harkiolaki
1Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK
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  • ORCID record for Maria Harkiolaki
  • For correspondence: maria.harkiolaki@diamond.ac.uk
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Abstract

Imaging of biological matter across resolution scales presents the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. We present here a correlative imaging platform developed specifically for imaging cells in 3D, under cryogenic conditions. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localisation methods are by and large diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly, platform for 3D correlative imaging of cells in cryo-preserved states using super-resolution structured illumination microscopy (SIM) in conjunction with soft X-ray tomography (SXT). The power of this new approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying novel virus-induced structures.

Footnotes

  • ↵± Joint last authors for methods

  • ↵+ Joint last authors for reovirus biology

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Posted January 14, 2020.
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Correlative cryo-structured illumination fluorescence microscopy and soft X-ray tomography elucidates reovirus intracellular release pathway
Ilias Kounatidis, Megan L Stanifer, Michael A. Phillips, Perrine Paul-Gilloteaux, Xavier Helligenstein, Hongchang Wang, Chidinma A. Okolo, Thomas M. Fish, Matthew C. Spink, David I. Stuart, Ilan Davis, Steeve Boulant, Jonathan M. Grimes, Ian M. Dobbie, Maria Harkiolaki
bioRxiv 2020.01.13.904623; doi: https://doi.org/10.1101/2020.01.13.904623
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Correlative cryo-structured illumination fluorescence microscopy and soft X-ray tomography elucidates reovirus intracellular release pathway
Ilias Kounatidis, Megan L Stanifer, Michael A. Phillips, Perrine Paul-Gilloteaux, Xavier Helligenstein, Hongchang Wang, Chidinma A. Okolo, Thomas M. Fish, Matthew C. Spink, David I. Stuart, Ilan Davis, Steeve Boulant, Jonathan M. Grimes, Ian M. Dobbie, Maria Harkiolaki
bioRxiv 2020.01.13.904623; doi: https://doi.org/10.1101/2020.01.13.904623

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