Segmented flow generator for serial crystallography at the European X-ray free electron laser

Abstract

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.

Potential of Time-Resolved Serial Femtosecond Crystallography Using High Repetition Rate XFEL Sources

“…Future methods also need to be compatible with sub-micrometer crystals, i.e., they must exhibit a low solvent background. Most of these rely on the synchronization of the sample delivery with the arrival of the X-ray pulse either by bunching the crystals (optical bunching [46]), injecting an alternative liquid during the dark time (segmented flow droplet injection [45]) or aiming to reduce the sample amount needed for a stable jet [38,44] (see Figure 1).…”

“…The segmented flow droplet injection (Figure 1c) is one approach that can be used to reduce sample wastage in SFX experiments and has already been tested at the European XFEL [45]. It aims to generate sub-nanoliter crystal suspension droplets embedded in an immiscible oil, allowing for injection with a traditional GDVN.…”

“…On the other hand, it is also necessary to consider testing the sample delivery devices for their photon-resistance properties before using them for pump-probe TR-SFX experiments in cases when the illumination of the samples in the nozzle is required. This can be achieved by the visual inspection of the fabricated devices after illumination with a range of optical laser powers using a similar laser setup to that of the pump-probe TR-SFX approach [44,45].…”

“…Figure 1. Schematic representations of standard (a,b) and innovative (c-e) injection delivery methods that are compatible with megahertz operation at the EuXFEL: (a) a GDVN-based liquid jet; (b) a DFFN-based liquid jet[38]; (c) a co-flow injection[44]; (d) a segmented flow droplet injection[45]; (e) a GDVN-based liquid jet with optical bunching[46].…”

ARIA—A VUV Beamline for EuPRAXIA@SPARC_LAB

“…Concerning the sample delivery, the injection of both liquid jets and aerosols will be possible. For liquid jets, different kinds of micro-jets, based on the gas dynamic virtual nozzle technology described in [56], including those developed for sample mixing [57] and for a lower sample consumption at pulsed sources [58], will be available. Molecules in the gas phase will instead be injected using an aerosol source, such as the one described in [59].…”