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Abstract Sample consumption for serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) remains a major limitation preventing broader use of this powerful technology in macromolecular crystallography. This drawback is exacerbated in the case of time-resolved (TR)-SFX experiments, where the amount of sample required per reaction time point is multiplied by the number of time points investigated. Thus, in order to reduce the limitation of sample consumption, here we demonstrate the implementation of segmented droplet generation in conjunction with a mix-and-inject approach for TR studies on NAD(P)H:quinone oxidoreductase 1 (NQO1). We present the design and application of mix-and-inject segmented droplet injectors for the Single Particles, Clusters, and Biomolecules & Serial Femtosecond Crystallography (SPB/SFX) instrument at the European XFEL (EuXFEL) with a synchronized droplet injection approach that allows liquid phase protein crystal injection. We carried out TR-crystallography experiments with this approach for a 305 ms and a 1190 ms time point in the reaction of NQO1 with its coenzyme NADH. With this successful TR-SFX approach, up to 97% of the sample has been conserved compared to continuous crystal suspension injection with a gas dynamic virtual nozzle. Furthermore, the obtained structural information for the reaction of NQO1 with NADH is an important part of the future elucidation of the reaction mechanism of this crucial therapeutic enzyme. 

Pump–probe experiments at X-ray free-electron laser (XFEL) facilities are a powerful tool for studying dynamics at ultrafast and longer timescales. Observing the dynamics in diverse scientific cases requires optical laser systems with a wide range of wavelength, flexible pulse sequences and different pulse durations, especially in the pump source. Here, the pump–probe instrumentation available for measurements at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of the European XFEL is reported. The temporal and spatial stability of this instrumentation is also presented. 

Abstract The emergence of high repetition-rate X-ray free-electron lasers (XFELs) powered by superconducting accelerator technology enables the measurement of significantly more experimental data per day than was previously possible. The European XFEL is expected to provide 27,000 pulses per second, over two orders of magnitude more than any other XFEL. The increased pulse rate is a key enabling factor for single-particle X-ray diffractive imaging, which relies on averaging the weak diffraction signal from single biological particles. Taking full advantage of this new capability requires that all experimental steps, from sample preparation and delivery to the acquisition of diffraction patterns, are compatible with the increased pulse repetition rate. Here, we show that single-particle imaging can be performed using X-ray pulses at megahertz repetition rates. The results obtained pave the way towards exploiting high repetition-rate X-ray free-electron lasers for single-particle imaging at their full repetition rate.