LAUSR

Volume Electron Microscopy (EM) not only enables dense reconstructions of neuronal circuits but provides the tissue context for targeted high resolution visualization of selected biological structures. Currently, 3D ultrastructure can be solved by destructive techniques including serial block-face electron microscopy (SB-SEM Denk & Horstmann, 2004; ), focused ion beam-scanning electron microscopy (FIBSEM (Heymann et al., 2006)) or serial sectioning (automated tape-collecting ultramicrotomy ATUM (K. J. Hayworth et al., 2014), transmission electron microscope camera array TEMCA (Bock et al., 2011)) methods. While destructive methods benefit from high alignment accuracy, they lack the option of reacquisition and hierarchical imaging. However, as microtomy-based approaches are limited by their poor z resolution, ion milling techniques are required if isotropic high resolution voxels are needed. Despite recent advances in the application of alternative milling strategies, targeted FIB-SEM imaging is still required to restrict the acquisition volume. This is mainly achieved by correlated workflows involving targeted trimming guided by endogenous and artificial landmarks (Bishop et al., 2011). X-ray micro computed tomography (microCT) (Bushong et al., 2015) has emerged as a tool for facilitated ROI relocation within the processed EM sample. So far, microCT imaging options are not commonly accessible and the technique only provides a virtual map for subsequent guided destructive sample preparation. An alternative prescreening of embedded tissue at a larger scale is implemented by rendering it accessible to light and electron imaging modalities. Ultrathick sectioning at 20 μm by the hot knife method provides samples that are accessible to large-scale FIB-SEM (Kenneth J. Hayworth et al., 2015) enabling seamless reconstruction of large tissue blocks.