RT Journal Article SR Electronic T1 Plasma FIB milling for the determination of structures in situ JF bioRxiv FD Cold Spring Harbor Laboratory SP 2022.08.01.502333 DO 10.1101/2022.08.01.502333 A1 Casper Berger A1 Maud Dumoux A1 Thomas Glen A1 Neville B.-y. Yee A1 John M. Mitchels A1 Zuzana Patáková A1 James H Naismith A1 Michael Grange YR 2022 UL http://biorxiv.org/content/early/2022/08/01/2022.08.01.502333.abstract AB Structural biology inside cells and tissues requires methods able to thin vitrified specimens to electron transparent thicknesses. Until now, focused ions beams based on gallium have been used. However, ion implantation, changes to surface chemistry and an inability to access high currents limit Gallium as an ion beam source. Here, we show that plasma-coupled ion sources can produce cryogenic lamella of vitrified human cells in a robust and automated manner, with quality sufficient for pseudo-atomic structure determination. In addition, these lamellae were produced in a prototype microscope equipped for long cryogenic run times (>1 week) and with multi-specimen support fully compatible with modern-day transmission electron microscopes. We demonstrate for the first time that plasma ion sources can be used for structural biology within cells, determining a structure in-situ to 4.9 Å and describing a workflow upon which different plasmas can be examined to streamline lamella fabrication further.Competing Interest StatementThe authors have declared no competing interest.