The reduction of FIB damage on cryo-lamella by lowering energy of ion beam revealed by a quantitative analysis

Qi Yang; Chunling Wu; Dongjie Zhu; Junxi Li; Jing Cheng; Xinzheng Zhang

doi:10.1016/j.str.2023.07.002

The reduction of FIB damage on cryo-lamella by lowering energy of ion beam revealed by a quantitative analysis

Structure. 2023 Oct 5;31(10):1275-1281.e4. doi: 10.1016/j.str.2023.07.002. Epub 2023 Jul 31.

Authors

Qi Yang¹, Chunling Wu², Dongjie Zhu², Junxi Li¹, Jing Cheng², Xinzheng Zhang³

Affiliations

¹ National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences (CAS), Beijing 100101, P.R. China; University of Chinese Academy of Sciences, Beijing 100049, P.R. China.
² National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences (CAS), Beijing 100101, P.R. China.
³ National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences (CAS), Beijing 100101, P.R. China. Electronic address: xzzhang@ibp.ac.cn.

PMID: 37527655
DOI: 10.1016/j.str.2023.07.002

Abstract

Focused ion beam (FIB) is widely used for thinning frozen cells to produce lamellae for cryo-electron microscopy imaging and for protein structures study in vivo. However, FIB damages the lamellae and a quantitative experimental analysis of the damage is lacking. We used a 30-keV gallium FIB to prepare lamellae of a highly concentrated icosahedral virus sample. The viruses were grouped according to their distance from the surface of lamellae and reconstructed. Damage to the approximately 20-nm-thick outermost lamella surface was similar to that from exposure to 16 e^-/Å² in a 300-kV cryo-electron microscope at high-resolution range. The damage was negligible at a depth beyond 50 nm, which was reduced to 30 nm if 8-keV Ga⁺ was used during polishing. We designed extra steps in the reconstruction refinement to maximize undamaged signals and increase the resolution. The results demonstrated that low-energy beam polishing was essential for high-quality thinner lamellae.

Keywords: cryo-EM; cryo-FIB damage; cryo-lamellae preparation; focused ion beam; low-energy ion milling.