Cryo-electron microscopy (Cryo-EM) has transformed structural biology by enabling near-atomic resolution of biomolecules in their native state. However, conventional sample preparation methods—pipetting, blotting, and plunge-freezing—require several seconds, obscuring the dynamics of rapid conformational changes. Recent time-resolved approaches, such as microfluidic mixing and spraying, have improved temporal resolution to tens of milliseconds but remain insufficient for probing sub-millisecond processes. To address this challenge, we developed a nanofluidic cryo-snapshot chip that enables time-resolved cryo-EM with sub-millisecond resolution. The device features nanochannels (∼200 nm depth) sandwiched between silicon nitride (SiN) windows (∼50 nm). Within these channels, biomolecular reactions are initiated by fluid mixing and evolve along downstream. By plunge-freezing the chip into cryogen, dynamic processes are preserved as a spatial sequence along the channel for TEM imaging. Temporal resolution can be tuned by adjusting flow velocities, providing access to transient intermediate states previously hidden from conventional methods. Here, we present the fabrication process of the nanofluidic chip and demonstrate its feasibility for capturing rapid biomolecular dynamics, establishing a framework for next-generation time-resolved cryo-EM.
Yoo et al. (Sun,) studied this question.