Multishot tomography for high-resolution in situ subtomogram averaging

[Display omitted] •• Beam Image-Shift (BIS) based imaging enables multishot tomogram acquisition along the tilt axis.•• Sample throughput is significantly increased.•• Positions are stable and result in high-quality tomograms.•• Subtomogram averaging of particles from in situ E. coli lamellas results in sub-nanometer resolution ribosome averages. Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) can resolve protein complexes at near atomic resolution, and when combined with focused ion beam (FIB) milling, macromolecules can be observed within their native context. Unlike single particle acquisition (SPA), cryo-ET can be slow, which may reduce overall project throughput. We here propose a fast, multi-position tomographic acquisition scheme based on beam-tilt corrected beam-shift imaging along the tilt axis, which yields sub-nanometer in situ STA averages.