Towards light-coupled sample preparation for time-resolved cryoEM studies.

The dynamic nature of protein and macromolecular complexes means that the capture of multiple sequential states along a reaction pathway can provide much greater insight into function than that obtained from a single static structure. We present a set of modular, easy-to-implement tools and workflows for optical excitation, on-grid characterization and tightly coupled rapid vitrification, establishing a proof-of-principle framework for time-resolved cryoEM and cryo-electron tomography (cryoET). We apply this framework to E. coli chemotaxis, in which serine-sensitive chemoreceptors initiate signalling upon ligand binding and undergo critical conformational changes within the chemosensory arrays. Using DMNB-caged serine [O-(4,5-dimethoxy-2-nitrobenzyl)-L-serine] as a model trigger, we quantified its photophysical properties and uncaging efficiency using UV-Vis spectroscopy and two-dimensional gas chromatography mass spectrometry (GC×GC-MS). Coupling a femtosecond-pulsed laser to a Vitrobot enabled reproducible reaction-to-vitrification delays of ∼150 ms, yielding intact E. coli minicells with well-preserved chemotaxis arrays suitable for in situ structural analysis by cryoET. This integrated approach provides a robust and generalisable framework for millisecond time-resolved cryoET, laying the groundwork for capturing transient conformational states in their native cellular context.