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