Modular microfluidics enables kinetic insight from time-resolved cryo-EM.

Mechanistic understanding of biochemical reactions requires structural and kinetic characterization of the underlying chemical processes. However, no single experimental technique can provide this information in a broadly applicable manner and thus structural studies of static macromolecules are often complemented by biophysical analysis. Moreover, the common strategy of utilizing mutants or crosslinking probes to stabilize intermediates is prone to trapping off-pathway artefacts and precludes determining the order of molecular events. Here we report a time-resolved sample preparation method for cryo-electron microscopy (trEM) using a modular microfluidic device, featuring a 3D-mixing unit and variable delay lines that enables automated, fast, and blot-free sample vitrification. This approach not only preserves high-resolution structural detail but also substantially improves sample integrity and protein distribution across the vitreous ice. We validate the method by visualising reaction intermediates of early RecA filament growth across three orders of magnitude on sub-second timescales. The trEM method reported here is versatile, reproducible, and readily adaptable to a broad spectrum of fundamental questions in biology.