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J Cell Sci. 2017 Jan 1;130(1):278-291. doi: 10.1242/jcs.188433. Epub 2016 Jul 21.

3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy.

Author information

1
Electron Microscopy Science Technology Platform, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
2
Host-pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, Mill Hill, London NW7 1AA, UK.
3
MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK.
4
The Babraham Institute, Cambridge CB22 3AT, UK.
5
Electron Microscopy Science Technology Platform, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK lucy.collinson@crick.ac.uk.

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.

KEYWORDS:

3D correlative light and electron microscopy; Autophagy; Entosis; HIV-1; Mycobacterium tuberculosis; Serial blockface SEM

PMID:
27445312
PMCID:
PMC5394779
DOI:
10.1242/jcs.188433
[Indexed for MEDLINE]
Free PMC Article

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