ATUM-Tomo: A multi-scale approach to cellular ultrastructure by combined volume scanning electron microscopy and electron tomography

• DOI: 10.7554/elife.90565.1
• Publication Date: 2023-10-10T10:20:25Z
• Authors: Georg Kislinger; Gunar Fabig; Antonia Wehn; Lucia Rodriguez; Hanyi Jiang; Cornelia Niemann; Andrey S. Klymchenko; Nikolaus Plesnila; Thomas Misgeld; Thomas Müller-Rei...; Igor Khalin; Martina Schifferer

Like other volume electron microscopy approaches, Automated Tape Collecting Ultramicrotomy (ATUM) enables imaging of serial sections deposited on thick plastic tapes by scanning (SEM). However, ATUM is unique enabling hierarchical and thus efficient screening for target structures as needed e.g., correlated light microscopy. SEM tape can only access the section surface, thereby limiting axial resolution to typical size cellular vesicles, an order magnitude lower than acquired xy resolution. In contrast, serial-section tomography (ET), a transmission microscopy-based approach, yields isotropic voxels at full EM resolution, but requires deposition electron-permeant thin fragile monolayer films – making large libraries difficult prone loss. To combine strength both we developed ‘ATUM-Tomo’, hybrid method, where are first reversibly attached via dissolvable coating, after detached transferred ET-compatible films. Thus, ATUM-SEM semi-thick consecutive ET one selected combines SEM’s fast recognition coarse rendering capability with ET’s high-resolution visualizations multi-scale interrogation ultrastructure. As proof-of-principle, applied correlative ATUM-Tomo study ultrastructural features blood brain barrier (BBB) leakiness around microthrombi in mouse model traumatic injury. Microthrombi associated sites BBB were identified confocal injected fluorescent electron-dense nanoparticles, then relocalized ATUM-SEM, finally interrogated ATUM-Tomo, workflow which created seamless zoom-in structural pathology from micro- nanometer scale. Overall, our new approach will substantially advance analysis biological phenomena that require cell- tissue-level contextualization finest subcellular textures.