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We introduce a hardware–software system for rapidly characterizing liquid microjets for x-ray diffraction experiments. An open-source python-based software package allows for programmatic and automated data collection and analysis. We show how jet speed, length, and diameter are influenced by nozzle geometry, gas flow rate, liquid viscosity, and liquid flow rate. We introduce “jet instability” and “jet probability” metrics to help quantify the suitability of a given nozzle for x-ray diffraction experiments. Among our observations were pronounced improvements in jet stability and reliability when using asymmetric needle-tipped nozzles, which allowed for the production of microjects smaller than 250 nm in diameter, traveling faster than 120 m/s. 

Gas-dynamic virtual nozzles (GDVNs) play a vital role in delivering biomolecular samples during diffraction measurements at X-ray free-electron laser facilities. Recently, submicrometer resolution capabilities of two-photon polymerization 3D printing techniques opened the possibility to quickly fabricate gas-dynamic virtual nozzles with practically any geometry. In our previous work, we exploited this capability to print asymmetric gas-dynamic virtual nozzles that outperformed conventional symmetric designs, which naturally leads to the question of how to identify the optimal gas-dynamic virtual nozzle geometry. In this work, we develop a 3D computational fluid dynamics pipeline to investigate how the characteristics of microjets are affected by gas-dynamic virtual nozzle geometry, which will allow for further geometry optimizations and explorations. We used open-source software (OpenFOAM) and an efficient geometric volume-of-fluid method ( isoAdvector ) to affordably and accurately predict jet properties for different nozzle geometries. Computational resources were minimized by utilizing adaptive mesh refinement. The numerical simulation results showed acceptable agreement with the experimental data, with a relative error of about 10% for our test cases that compared bell- and cone-shaped sheath-gas cavities. In these test cases, we used a relatively low sheath gas flow rate (6 mg/min), but future work including the implementation of compressible flows will enable the investigation of higher flow rates and the study of asymmetric drip-to-jet transitions. 

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.