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Publication Detail
3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy.
  • Publication Type:
    Journal article
  • Publication Sub Type:
    Article
  • Authors:
    Russell MR, Lerner TR, Burden JJ, Nkwe DO, Pelchen-Matthews A, Domart MC, Durgan J, Weston A, Jones ML, Peddie CJ, Carzaniga R, Florey O, Marsh M, Gutierrez MG, Collinson LM
  • Publication date:
    21/07/2016
  • Journal:
    Journal of cell science
  • Medium:
    Print-Electronic
  • Print ISSN:
    0021-9533
  • Language:
    eng
  • Addresses:
    Electron Microscopy Science Technology Platform, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, 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/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.
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