Xenon plasma focused ion beam lamella fabrication on high-pressure frozen specimens for structural cell biology

Abstract

Cryo focused ion beam lamella preparation is a potent tool forin situstructural biology, enabling the study of macromolecules in their native cellular environments. However, throughput is currently limited, especially for thicker, more biologically complex samples. We describe how xenon plasma focused ion beam milling can be used for routine bulk milling of thicker, high-pressure frozen samples during lamellae preparation with a high success rate and determine a 4.0 Å structure of theEscherichia coliribosome on these lamellae using sub volume averaging. We determine the effects of increased ion currents on sample integrity during bulk milling of thicker planar samples, also showing that beyond an initial region of damage, no measurable structural damage propagates beyond this. The use of xenon results in substantial structural damage to particles up to 30 nm in depth from the milled surfaces, with detectable damage observed to 45 nm. Ours results outlines how the use of high currents using xenon plasma focused ion beam milling may be integrated into FIB milling regimes for preparing thin lamellae for high-resolutionin situstructural biology.