278.full.pdf (24.22 MB)

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

Download (24.22 MB)
journal contribution
posted on 15.10.2020, 13:31 by Matthew RG Russell, Thomas R Lerner, Jemima J Burden, David O Nkwe, Annegret Pelchen-Matthews, Marie-Charlotte Domart, Joanne Durgan, Anne Weston, Martin L Jones, Christopher J Peddie, Raffaella Carzaniga, Oliver Florey, Mark Marsh, Maximiliano G Gutierrez, Lucy M Collinson
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.