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.
| Original language | English |
|---|---|
| Pages (from-to) | 278-291 |
| Number of pages | 14 |
| Journal | Journal of Cell Science |
| Volume | 130 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - Jan 1 2017 |
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All Science Journal Classification (ASJC) codes
- Cell Biology
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3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy. / Russell, Matthew R.G.; Lerner, Thomas R.; Burden, Jemima J.; Nkwe, David O.; Pelchen-Matthews, Annegret; Domart, Marie Charlotte; Durgan, Joanne; Weston, Anne; Jones, Martin L.; Peddie, Christopher J.; Carzaniga, Raffaella; Florey, Oliver; Marsh, Mark; Gutierrez, Maximiliano G.; Collinson, Lucy M.
In: Journal of Cell Science, Vol. 130, No. 1, 01.01.2017, p. 278-291.Research output: Contribution to journal › Article
TY - JOUR
T1 - 3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy
AU - Russell, Matthew R.G.
AU - Lerner, Thomas R.
AU - Burden, Jemima J.
AU - Nkwe, David O.
AU - Pelchen-Matthews, Annegret
AU - Domart, Marie Charlotte
AU - Durgan, Joanne
AU - Weston, Anne
AU - Jones, Martin L.
AU - Peddie, Christopher J.
AU - Carzaniga, Raffaella
AU - Florey, Oliver
AU - Marsh, Mark
AU - Gutierrez, Maximiliano G.
AU - Collinson, Lucy M.
PY - 2017/1/1
Y1 - 2017/1/1
N2 - 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.
AB - 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.
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U2 - 10.1242/jcs.188433
DO - 10.1242/jcs.188433
M3 - Article
VL - 130
SP - 278
EP - 291
JO - Journal of Cell Science
JF - Journal of Cell Science
SN - 0021-9533
IS - 1
ER -