Thinner is not always better: Optimizing cryo-lamellae for subtomogram averaging

Abstract

Cryo–electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyze biomolecules in situ by subtomogram averaging, yet data quality critically depends on specimen thickness. Cells that are too thick for transmission imaging can be thinned into lamellae by cryo–focused ion beam (cryo-FIB) milling. Despite being a crucial parameter directly affecting attainable resolution, optimal lamella thickness has not been systematically investigated nor the extent of structural damage caused by gallium ions used for FIB milling. We thus systematically determined how resolution is affected by these parameters. We find that ion-induced damage does not affect regions more than 30 nanometers from either lamella surface and that up to ~180-nanometer lamella thickness does not negatively affect resolution. This shows that there is no need to generate very thin lamellae and lamella thickness can be chosen such that it captures cellular features of interest, thereby opening cryo-ET also for studies of large complexes.

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

“…4d,e). The limit of the depth of damage is comparable to other studies using argon (Berger et al, 2023; Chaillet et al, 2023; Parkhurst et al, 2023) and gallium beams (Lucas and Grigorieff, 2023; Yang et al, 2023; Tuijtel et al, 2024), despite notable differences in methodology, each of which have relative merits. Regardless of the technique used, it is important to correct for variations in factors such as local lamella thickness, motion and charging.…”

“…Using in situ subtomogram averaging and B-factor analysis (Rosenthal and Henderson, 2003), it was demonstrated that 30 kV argon PFIB milling on biological samples results in damage characterised by reduced information content at depths up to 30 to 45 nm from the milling surfaces (Berger et al, 2023). The damage layer for 8 kV and 30 kV gallium FIB milling has been characterised (Lucas and Grigorieff, 2023;Yang et al, 2023;Tuijtel et al, 2024), reporting penetration depths for the damage between 30 and 60 nm, with reduced depth of damage at lower voltage FIB milling. We present a workflow for the use of xenon PFIB milling to prepare lamellae from ~25 µm thick highpressure frozen biological samples with currents up to 60 nA.…”