title: 3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy.
abstract: The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research.
authors.affiliation: Electron Microscopy Science Technology Platform, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
authors.forename: Matthew R G
authors.identifier: 0000-0003-4608-7669
authors.initials: MR
authors.lastname: Russell
authors.affiliation: Host-pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, Mill Hill, London NW7 1AA, UK.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Lerner
authors.affiliation: MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK.
authors.forename: Jemima J
authors.initials: JJ
authors.lastname: Burden
authors.affiliation: MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK.
authors.forename: David O
authors.initials: DO
authors.lastname: Nkwe
authors.affiliation: MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK.
authors.forename: Annegret
authors.initials: A
authors.lastname: Pelchen-Matthews
authors.affiliation: Electron Microscopy Science Technology Platform, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
authors.forename: Marie-Charlotte
authors.initials: MC
authors.lastname: Domart
authors.affiliation: The Babraham Institute, Cambridge CB22 3AT, UK.
authors.forename: Joanne
authors.initials: J
authors.lastname: Durgan
authors.affiliation: Electron Microscopy Science Technology Platform, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
authors.forename: Anne
authors.initials: A
authors.lastname: Weston
authors.affiliation: Electron Microscopy Science Technology Platform, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
authors.forename: Martin L
authors.initials: ML
authors.lastname: Jones
authors.affiliation: Electron Microscopy Science Technology Platform, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Peddie
authors.affiliation: Electron Microscopy Science Technology Platform, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Carzaniga
authors.affiliation: The Babraham Institute, Cambridge CB22 3AT, UK.
authors.forename: Oliver
authors.initials: O
authors.lastname: Florey
authors.affiliation: MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Marsh
authors.affiliation: Host-pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, Mill Hill, London NW7 1AA, UK.
authors.forename: Maximiliano G
authors.initials: MG
authors.lastname: Gutierrez
authors.affiliation: Electron Microscopy Science Technology Platform, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK lucy.collinson@crick.ac.uk.
authors.forename: Lucy M
authors.identifier: 0000-0003-0260-613X
authors.initials: LM
authors.lastname: Collinson
mesh_terms: D002470:Cell Survival; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000382:microbiology / Q000648:ultrastructure*; D057691:Entosis; D006678:HIV / Q000648:ultrastructure; D006801:Humans; D021621:Imaging, Three-Dimensional; D042541:Intracellular Space / Q000382:microbiology; D008264:Macrophages / Q000648:ultrastructure* / Q000821:virology; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D009000:Monocytes / Q000166:cytology; D009169:Mycobacterium tuberculosis / Q000254:growth & development / Q000648:ultrastructure
keywords: 3D correlative light and electron microscopy; Autophagy; Entosis; HIV-1; Mycobacterium tuberculosis; Serial blockface SEM