Neuropathology on tape – applications of ATUM-based volume EM

Abstract text (incl. figure legends and references)

Volume Electron Microscopy (vEM) reveals biological structures at nanometer resolution in three dimensions and resolves ambiguities of two-dimensional representations. However, as the structures of interest – like disease hallmarks emerging from neuropathology – are often rare but the field of view is small, this can easily turn a vEM project into a needle in a haystack problem. One solution for this is correlated light and electron microscopy (CLEM), which requires precise coordinate transfer between the two imaging modalities. With array tomography methods, serial ultrathin sections are collected into a storable tissue library, thus restoring precious samples like human biopsies and enabling repetitive imaging at different resolution levels for an SEM-based search strategy. For this, automated tape collecting ultramicrotomy (ATUM) has been developed to reliably collect serial ultrathin sections via a conveyor belt onto a plastic tape that is later mounted onto silicon wafers for serial scanning EM (SEM) (Kasthuri, Hayworth et al., 2015). The ATUM-SEM procedure is highly modular and enables targeting and high resolution imaging of specific structures. The variation of section thickness determines axial resolution and screening speed. We have developed ATUM hybrid methods that enable both, fast screening and targeted imaging at isotropic voxels (Kislinger, Gnägi et al., 2020). We exemplify targeted ATUM-SEM in mouse models for the reconstruction of Alzheimer"s disease plaques and for the visualization of Blood Brain Barrier leakage (Khalin, Adarsh et al., 2022). In order to correlate transcript levels with ultrastructure, we designed an indirect correlative method called SPcEM which combines ATUM-SEM with Spatial Transcriptomics (Androvic, Schifferer et al., 2022). In a mouse demyelination model adjacent cryosections are inspected by either of the two methods for sophisticated microglia classification.

Androvic P, Schifferer M, Anderson KP, Cantuti-Castelvetri L, Ji H, Liu L, Besson-Girard S, Knoferle J, Simons M, Gokce O (2022) Spatial Transcriptomics-correlated Electron Microscopy. bioRxiv: 2022.05.18.492475

Khalin I, Adarsh N, Schifferer M, Wehn A, Groschup B, Misgeld T, Klymchenko A, Plesnila N (2022) Size-Selective Transfer of Lipid Nanoparticle-Based Drug Carriers Across the Blood Brain Barrier Via Vascular Occlusions Following Traumatic Brain Injury. Small (Weinheim an der Bergstrasse, Germany) n/a: 2200302

Kislinger G, Gnägi H, Kerschensteiner M, Simons M, Misgeld T, Schifferer M (2020a) ATUM-FIB microscopy for targeting and multiscale imaging of rare events in mouse cortex. STAR Protocols 1: 100232Kislinger G, Gnägi H, Kerschensteiner M, Simons M, Misgeld T, Schifferer M (2020b) Multiscale ATUM-FIB Microscopy Enables Targeted Ultrastructural Analysis at Isotropic Resolution. iScience 23: 101290

Fig. 1 ATUM-SEM and ATUM-FIB of AD plaques

Fig. 2 BBB-CLEM with fluorescent and gold nanoparticles

Fig. 3 SPcEM in demyelinating disease