Time-resolved cryo-EM captures short-lived molecular states on ms timescales.

Cryo-ET allows to study biomolecular structures in their physiological environments.

Cryo-CLEM increases throughput by locating target molecules within the specimen.

Cryo-FIB milling thins specimens to visualize previously inaccessible cellular areas.

All steps of cryogenic electron-microscopy (cryo-EM) workflows have rapidly evolved over the last decade. Advances in both single-particle analysis (SPA) cryo-EM and cryo-electron tomography (cryo-ET) have facilitated the determination of high-resolution biomolecular structures that are not tractable with other methods. However, challenges remain. For SPA, these include improved resolution in an additional dimension: time. For cryo-ET, these include accessing difficult-to-image areas of a cell and finding rare molecules. Finally, there is a need for automated and faster workflows, as many projects are limited by throughput. Here, we review current developments in SPA cryo-EM and cryo-ET that push these boundaries. Collectively, these advances are poised to propel our spatial and temporal understanding of macromolecular processes.

© 2022 The Author(s). Published by Elsevier Ltd.