Purchase of electron microscopes for DANCEM

The requirements come from the scientific objectives for DANCEM that aims at making the infrastructure available for a range of research areas including neuroscience, genome editing, infection biology, cancer research, and drug discovery employing workflows in single particle analysis, microcrystal electron diffraction and cryogenic electron tomography. DANCEM will in particular focus on establishing new cryogenic electron tomography research workflows. The DANCEM user base covers research in cells, tissues, microbial communities, plant materials and materials science that need the essential requirements described below.

The DANCEM consortium already operates TFS instruments: Titan Krios G2, Glacios and Hydra Bio at UCPH; Titan Krios G3i, Titan Krios G1 and Aquilos 2 at AU. • R1 Lamellae must be transferred from the cryo-PFIB to the cryo-TEM under vacuum, autoloader to autoloader, in a closed capsule holding a cassette of up to 12 autogrids, without exposure to atmosphere or open liquid nitrogen.

This protects the lamellae from ice contamination and devitrification, which directly decides the yield and quality of the cryoET research. • R2 The new cryo-TEMs and cryo-PFIBs must use the same autogrid format as the six existing instruments, and the same cassette and capsule as the existing autoloader instruments, so that grids and lamellae can move between any old and new instrument, at either node, without re-clipping or re-mounting, and can be retrieved in the vitreous state. • R3 The new instruments must use the same TFS acquisition, automation and correlative software environment as the existing instruments (EPU, Tomography, MAPS correlative projects shared between TEM, SEM and FIB, and AutoTEM Cryo/WebUI for lamella preparation), so that targets, atlases, settings and trained procedures carry over between instruments and nodes. • R4 The AU node needs a cryo-PFIB for thick samples and tissues with four selectable ion species (Xe, Ar, O, N) switched in software, integrated cryo lift-out from autogrids and 3 and 6 mm high-pressure-freezing planchettes, and volume EM at cryo and room temperature.

The existing gallium-ion Aquilos 2 is not well suited to thicker tissue-based samples. Published comparisons show that milling quality depends on the ion species used (Dumoux et al., eLife 2023) and besides Xenon and Argon, availability of Oxygen for biological samples is essential for the new workflows. • R5 Each node needs a 120 kV screening TEM that runs the same EPU, Tomography 5 and Maps 3 software as the Krios 5, so that screening results carry directly into high-end data collection.