biostudies-literatureBhattacharjee SNational Institutes of HealthNIGMS NIH HHS782-796.e23https://www.ebi.ac.uk/biostudies/studies/S-EPMC10872292biostudies-literatureUnknown187(3)The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here, we introduce a time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency. Coating of PDMS walls with SiO₂ virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1,000 ms, the device allows us to follow in vitro reactions of biological molecules at resoCellTime resolution in cryo-EM using a PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling.PMC10872292R35GM139453R35 GM139453Dadhwal PZhang ZFeng XMaji SFrank JBrown ZPBhattacharjee SfalseTime resolution in cryo-EM using a PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling.The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here, we introduce a time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency. Coating of PDMS walls with SiO₂ virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1,000 ms, the device allows us to follow in vitro reactions of biological molecules at reso2024-01-01T00:00:00Z2024 Feb2026-06-02T11:58:50.888Z2025-04-03T23:22:48.224ZS-EPMC108722923824454710.1016/j.cell.2023.12.027