Multiscale ATUM-FIB microscopy enables targeted ultrastructural analysis at isotropic resolution

Volume electron microscopy enables the ultrastructural analysis of biological tissues and is essential for dense reconstructions e.g. of neuronal circuits. So far, three-dimensional analysis is based on either serial sectioning followed by sequential imaging (ATUM, ssTEM/SEM) or serial block-face imaging (SB-SEM, FIB-SEM), where imaging is intercalated with sectioning. Currently, the techniques involving ultramicrotomy allow scanning large fields of view, but afford only limited z-resolution determined by section thickness, while ion beam-milling approaches yield isotropic voxels, but are restricted in volume size. Now we present a hybrid method, named ATUM-FIB, which combines the advantages of both approaches: ATUM-FIB is based on serial sectioning of tissue into semithick (2-10 µm) resin sections that are collected onto transparent tape. 3D information obtained by serial light and electron microscopy allows identifying regions of interest that are then directly accessible for targeted FIB-SEM. The set of serial semithin sections thus represent a tissue ‘library’, which provides information about microscopic tissue context that can then be probed ‘on demand’ by local high resolution analysis. We demonstrate the potential of this technique to reveal the ultrastructure of rare but pathologically important events by identifying microglia contact sites with amyloid plaques in a mouse model for familial Alzheimer’s disease.