Structural biology studies inside cells and tissues require methods to thin vitrified specimens to electron transparency. Until now, focused ion beams based on gallium have been used. However, ion implantation, changes to surface chemistry and an inability to access high currents limit gallium application. Here, we show that plasma-coupled ion sources can produce cryogenic lamellae of vitrified human cells in a robust and automated manner, with quality sufficient for pseudo-atomic structure determination. 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 that plasma ion sources can be used for structural biology within cells, determining a structure in situ to 4.9 Å, and characterise the resolution dependence on particle distance from the lamella edge. We describe a workflow upon which different plasmas can be examined to further streamline lamella fabrication.

Artificial Intelligence and Informatics The Rosalind Franklin Institute Harwell Science and Innovation Campus Didcot OX11 0QS United Kingdom

Division of Structural Biology Wellcome Centre for Human Genetics University of Oxford OX3 7BN Oxford United Kingdom

Structural Biology The Rosalind Franklin Institute Harwell Science and Innovation Campus Didcot OX11 0QS United Kingdom

Thermo Fisher Scientific Brno s r o Brno 627 00 Czech Republic

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