ATUM-FIB microscopy for targeting and multiscale imaging of rare events in mouse cortex

Summary Here, we describe a detailed workflow for ATUM-FIB microscopy, a hybrid method that combines serial-sectioning scanning electron microscopy (SEM) with focused ion beam SEM (FIB-SEM). This detailed protocol is optimized for mouse cortex samples. The main processing steps include the generation of semi-thick sections from sequentially cured resin blocks using a heated microtomy approach. We demonstrate the different imaging modalities, including serial light and electron microscopy for target recognition and FIB-SEM for isotropic imaging of regions of interest. For complete details on the use and execution of this protocol, please refer to Kislinger et al. (2020) .