![]() In both cases, the revealed surface of the block is imaged using a backscattered electron (BSE) detector, and the process repeated sequentially to build up a stack of images through the volume of the sample. In FIB-SEM, a gallium ion beam sputters slices of material from the blockface, whereas in SBF-SEM a diamond knife in a miniaturised ultramicrotome removes thin slices from the blockface. In array tomography, sections are cut manually or automatically ( Hayworth et al., 2006) and placed in an array on a silicon wafer for large area imaging in the SEM. These innovative ‘volume electron microscopy’ techniques ( Kremer et al., 2015 Peddie and Collinson, 2014) include array tomography ( Micheva and Smith, 2007 Wacker and Schroeder, 2013), focused ion beam SEM (FIB-SEM) ( Heymann et al., 2006) and serial blockface SEM (SBF-SEM) ( Denk and Horstmann, 2004). However, automated systems based on the scanning electron microscopy (SEM) are gaining in popularity. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research.Įlectron microscopy for CLEM was traditionally performed by manually serial sectioning the sample and imaging each section using transmission electron microscopy (TEM), usually requiring more than 100 sections to image a single cell. ![]() 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. 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. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. 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 processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. ![]()
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