A simple and practical approach for protein serial crystallography using grease matrix and large-area support film.

Serial femtosecond crystallography (SFX) enables structural determination of biological macromolecules at room temperature using ultrashort X-ray pulses from an X-ray free-electron laser, thereby minimizing radiation damage and capturing dynamic structural changes. Efficient sample delivery is essential for collecting many diffraction patterns. The grease matrix injection method reduces sample consumption but may encounter nozzle clogging and unstable flow when the crystal size is large, the crystal density is high, or the viscosity of the crystallization buffer-grease mixture increases. Fixed-target approaches, in which crystals are dispersed on a support film, offer a stable alternative with lower sample consumption. Here, we present an optimized fixed-target system integrating a grease matrix and a large-area support film, enhancing sample stability, data collection efficiency, and structural resolution while enabling potential hydrogen atom detection in SFX experiments.