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

• DOI: 10.1126/sciadv.adk6285
• Publication Date: 2024-04-26T17:58:41Z
• Authors: Maarten W. Tuijtel; Sergio Cruz-León; Jan Philipp Kreysing; Sonja Welsch; Gerhard Hummer; Martin Beck; Beata Turoňová
• Keywords: Electron Microscope Tomography; 0303 health sciences; 03 medical and health sciences; Cryoelectron Microscopy; Image Processing, Computer-Assisted; Humans; Physical and Materials Sciences; Gallium

Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and 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 cryo-focused ion beam (cryo-FIB) milling. Despite being crucial parameter directly affecting attainable resolution, optimal lamella thickness has not been systematically investigated nor the extent of structural damage caused gallium ions used FIB We thus determined how resolution affected these parameters. find ion-induced does affect regions more than 30 nanometers from either surface up ~180-nanometer negatively resolution. This shows there no need generate very thin chosen such it captures cellular features interest, thereby opening cryo-ET also studies large complexes.