Strategies for time-resolved cryogenic electrontomography of transient cellular processes

Cryogenic electron tomography provides molecular-scale information from frozen-hydrated cells and tissues, with each tomogram representing a selected cellular volume at the time of vitrification. Time-resolved cryo-ET aims to build structural descriptions of cellular processes by relating vitrified states to defined perturbations or stages of transient cellular events. This requires the relevant state to be generated reproducibly by an experimental perturbation or identified reliably before tomography and sampled in sufficient numbers for comparison. Here, we give an overview of four experimental routes by which temporal information is introduced into cellular cryo-ET experiments, discuss the conditions under which each can provide reliable structural and temporal data, and identify interpretive challenges that are common across all approaches. Time-resolved cryo-ET is a developing area with considerable potential to provide mechanistic insights into transient cellular processes that remain difficult to access by other structural methods.