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Abstract Detecting microsecond structural perturbations in biomolecules has wide relevance in biology, chemistry and medicine. Here we show how MHz repetition rates at X-ray free-electron lasers can be used to produce microsecond time-series of protein scattering with exceptionally low noise levels of 0.001%. We demonstrate the approach by examining Jɑ helix unfolding of a light-oxygen-voltage photosensory domain. This time-resolved acquisition strategy is easy to implement and widely applicable for direct observation of structural dynamics of many biochemical processes. 

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. 

X-ray free-electron lasers (XFELs) can probe chemical and biological reactions as they unfold with unprecedented spatial and temporal resolution. A principal challenge in this pursuit involves the delivery of samples to the X-ray interaction point in such a way that produces data of the highest possible quality and with maximal efficiency. This is hampered by intrinsic constraints posed by the light source and operation within a beamline environment. For liquid samples, the solution typically involves some form of high-speed liquid jet, capable of keeping up with the rate of X-ray pulses. However, conventional jets are not ideal because of radiation-induced explosions of the jet, as well as their cylindrical geometry combined with the X-ray pointing instability of many beamlines which causes the interaction volume to differ for every pulse. This complicates data analysis and contributes to measurement errors. An alternative geometry is a liquid sheet jet which, with its constant thickness over large areas, eliminates the problems related to X-ray pointing. Since liquid sheets can be made very thin, the radiation-induced explosion is reduced, boosting their stability. These are especially attractive for experiments which benefit from small interaction volumes such as fluctuation X-ray scattering and several types of spectroscopy. Although their use has increased for soft X-ray applications in recent years, there has not yet been wide-scale adoption at XFELs. Here, gas-accelerated liquid sheet jet sample injection is demonstrated at the European XFEL SPB/SFX nano focus beamline. Its performance relative to a conventional liquid jet is evaluated and superior performance across several key factors has been found. This includes a thickness profile ranging from hundreds of nanometres to 60 nm, a fourfold increase in background stability and favorable radiation-induced explosion dynamics at high repetition rates up to 1.13 MHz. Its minute thickness also suggests that ultrafast single-particle solution scattering is a possibility. 

A 3D-printed modular droplet injector successfully delivered microcrystals of human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin with electrical stimulation in a serial crystallography experiment at 120 Hz repetition rate. 

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. 

Serial crystallography of membrane proteins often employs high-viscosity injectors (HVIs) to deliver micrometre-sized crystals to the X-ray beam. Typically, the carrier medium is a lipidic cubic phase (LCP) media, which can also be used to nucleate and grow the crystals. However, despite the fact that the LCP is widely used with HVIs, the potential impact of the injection process on the LCP structure has not been reported and hence is not yet well understood. The self-assembled structure of the LCP can be affected by pressure, dehydration and temperature changes, all of which occur during continuous flow injection. These changes to the LCP structure may in turn impact the results of X-ray diffraction measurements from membrane protein crystals. To investigate the influence of HVIs on the structure of the LCP we conducted a study of the phase changes in monoolein/water and monoolein/buffer mixtures during continuous flow injection, at both atmospheric pressure and under vacuum. The reservoir pressure in the HVI was tracked to determine if there is any correlation with the phase behaviour of the LCP. The results indicated that, even though the reservoir pressure underwent (at times) significant variation, this did not appear to correlate with observed phase changes in the sample stream or correspond to shifts in the LCP lattice parameter. During vacuum injection, there was a three-way coexistence of the gyroid cubic phase, diamond cubic phase and lamellar phase. During injection at atmospheric pressure, the coexistence of a cubic phase and lamellar phase in the monoolein/water mixtures was also observed. The degree to which the lamellar phase is formed was found to be strongly dependent on the co-flowing gas conditions used to stabilize the LCP stream. A combination of laboratory-based optical polarization microscopy and simulation studies was used to investigate these observations.