Combination of an inject-and-transfer system for serial femtosecond crystallography

Serial femtosecond crystallography (SFX) enables the determination of room-temperature crystal structures of macromolecules with minimized radiation damage and provides time-resolved molecular dynamics by pump–probe or mix-and-inject experiments. In SFX, a variety of sample delivery methods with unique advantages have been developed and applied. The combination of existing sample delivery methods can enable a new approach to SFX data collection that combines the advantages of the individual methods. This study introduces a combined inject-and-transfer system (BITS) method for sample delivery in SFX experiments: a hybrid injection and fixed-target scanning method. BITS allows for solution samples to be reliably deposited on ultraviolet ozone (UVO)-treated polyimide films, at a minimum flow rate of 0.5 nl min⁻¹, in both vertical and horizontal scanning modes. To utilize BITS in SFX experiments, lysozyme crystal samples were embedded in a viscous lard medium and injected at flow rates of 50–100 nl min⁻¹ through a syringe needle onto a UVO-treated polyimide film, which was mounted on a fixed-target scan stage. The crystal samples deposited on the film were raster scanned with an X-ray free electron laser using a motion stage in both horizontal and vertical directions. Using the BITS method, the room-temperature structure of lysozyme was successfully determined at a resolution of 2.1 Å, and thus BITS could be utilized in future SFX experiments.