Russell, MRG;
Lerner, TR;
Burden, JJ;
Nkwe, DO;
Pelchen-Matthews, A;
Domart, M-C;
Durgan, J;
... Collinson, LM; + view all
(2017)
3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy.
Journal of Cell Science
, 130
(1)
pp. 278-291.
10.1242/jcs.188433.
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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 monocytederived 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.
| Type: | Article |
|---|---|
| Title: | 3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1242/jcs.188433 |
| Publisher version: | http://dx.doi.org/10.1242/jcs.188433 |
| Language: | English |
| Additional information: | © 2017. Published by The Company of Biologists Ltd http://creativecommons.org/licenses/by/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. |
| Keywords: | Life Sciences & Biomedicine, Cell Biology, 3D correlative light and electron microscopy, Serial blockface SEM, Mycobacterium tuberculosis, Autophagy, HIV-1, Entosis, HIV-1 Assembly Compartments, Plasma-membrane, Array Tomography, Image Data, Mammalian-cells, In-vivo, Macrophages, Visualization, Ultrastructure, Tuberculosis |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Lab for Molecular Cell Bio MRC-UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > Institute for Global Health |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1509339 |
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