A PDMS-based Microfluidic Chip Assembly for Time-Resolved Cryo-EM (TRCEM) Sample Preparation.

Time-resolved cryo-EM (TRCEM) makes it possible to provide structural and kinetic information on a reaction of biomolecules before the equilibrium is reached. Several TRCEM methods have been developed in the past to obtain key insights into the mechanism of action of molecules and molecular machines on the time scale of tens to hundreds of milliseconds, which is unattainable by the normal blotting method. Here, we present our TRCEM setup utilizing a polydimethylsiloxane (PDMS)-based microfluidics chip assembly, comprising three components: a PDMS-based, internally SiO₂-coated micromixer, a glass-capillary microreactor, and a PDMS-based microsprayer for depositing the reaction product onto the EM grid. As we have demonstrated in recent experiments, this setup is capable of addressing problems of severe sample adsorption and ineffective mixing of fluids and leads to highly reproducible results in applications to the study of translation. As an example, we used our TRCEM sample preparation method to investigate the molecular mechanism of ribosome recycling mediated by High frequency of lysogenization X (HflX), which demonstrated the efficacy of the TRCEM device and its capability to yield biologically significant, reproducible information. This protocol has the promise to provide structural and kinetic information on pre-equilibrium intermediates in the 10-1,000 ms time range in applications to many other biological systems. Key features • Design and fabrication of high-performance splitting-and-recombination-based micromixer and planar microsprayer. • Protocol for SiO₂ coating on the PDMS surface and fabrication of the microfluidic chip assembly. • Preparation of time-resolved cryo-EM sample in the time range of 10-1,000 ms. • Data collection on EM grid covered with droplets from the microsprayer.