Interleaved multi-magnification cryo-electron tomography bridges cellular and structural biology

Abstract

Cryo-electron tomography (cryo-ET) enables in situ structural analysis of macromolecular complexes within native cellular environments. However, the limited field of view required for high-resolution structure determination necessarily restricts a wider assessment of the broader cellular context. We present a multi-magnification cryo-ET acquisition strategy that integrates low- and high-magnification information from coincident sample regions during the same tilt-series. By interleaving acquisition of the magnifications at each tilt angle, this strategy enables simultaneous collection of large field-of-view, low-resolution tomograms and high-resolution, small field-of-view tomograms while minimising the impact of the increased electron dose. We demonstrate that we can capture cellular organisation across tens of microns, while still enabling subtomogram averaging to resolutions below 4 [A]. This integrated acquisition framework establishes a practical route to multi-scale cryo-ET, bridging molecular and cellular scales for more comprehensive biological insight.