4th Liquid Sample Delivery Workshop @ EuXFEL

Liquid phase sample delivery at LCLS is highly standardized. Hutches use similar or identical EPICS based GUIs running common controls hardware. Liquid injectors for sheets, jets, and drops are mass produced in foundries with tight manufacturing tolerances. All chip-based nozzles fit the standard SLAC chip interface which is deployed throughout the endstations. Sample handling systems for recirculation operate in the ml/min range – a range in which sample consumption is independent of sample delivery and depends only on exposure to X-rays. So is there anything left to do? The current state of LCLS liquid sample delivery and future directions for hardware development will be presented.

Serial crystallography has emerged as a unique experimental technique for determining molecular structures in both static and dynamic states, utilizing synchrotron and XFEL sources. To date, numerous techniques have been developed to transport microcrystals while maintaining stability and appropriate environmental conditions, including liquid-jet injection, droplet-based delivery, and fixed-target scanning methods [1]. To address diverse experimental demands, we have established several unique sample delivery platforms, thereby improving sample utilization efficiency, environmental control, and overall experimental versatility for the SFX instrument at the NCI endstation of PAL-XFEL. These include PSD and CMD injectors for liquid jet and LCP delivery, as well as fixed target systems with one- and two-dimensional geometries [2].

This presentation will provide an overview of serial crystallography methodologies for the SFX instrument at PAL-XFEL and highlight the recent development of a negative-pressure-driven sample delivery system (NDS). It enables stable microcrystal delivery in crystallization solution over a wider range of flow rates and offers improved environmental control for crystals.

[1] A. Banari et al., Nat. Methods 22, 1420.
[2] Park, J., Nam, K. H., photonics 10, 557.

The aerosol sample delivery system available at the SPB/SFX instrument at the European XFEL and sample characterization methods prior to X-ray imaging are presented. The presentation covers the setup, recent examples and sample requirements.

Time-resolved X-ray crystallography using serial approaches has received increasing attention in recent years, not only at XFELs but also at synchrotron facilities. Beamline I24 at Diamond Light Source (UK) has been at the forefront of developments in instrumentation, data collection strategies and data processing, lowering the entry barrier to a wider user community. Although originally designed for conventional microfocus MX, I24 has evolved into a versatile beamline that supports serial approaches and multiple modes of room-temperature data collection. Currently, the beamline features a modular sample environment with a plug-and-play design, enabling pin-based cryo MX and serial experiments to be performed within the same shift, with changeover requiring only a few minutes. Serial experiments can be carried out on thin film arrays, fixed targets [1] or using a high viscosity extruder. Several methods for reaction initiation, including light activation, drop-on-chip mixing and the use of photocages, have been successfully applied to initiate reactions in micro-crystalline slurries for dynamic studies.

Recent developments on I24 will be presented including optimisation of fixed-targets for time-resolved SSX (and SFX [2]) on challenging targets requiring anaerobic or dark conditions, exploring the advantages of high energy data collection, new detectors, and complementary spectroscopic methods. Case studies demonstrating the use of nitric oxide [3] and oxygen [4] photocages for efficiently initiating time-dependent reactions will also be discussed.

[1] Owen, R. L., et al. (2017). Acta Cryst. D, 73, 373-378.
[2] Sherrell, D. A., et al. (2015). J. Synchrotron. Radiat., 22, 1372-1378.
[3] Smyth, P., et al. (2025). IUCrJ, 12, 582-594.
[4] Jaho, S., et al. (2026). bioRxiv, 2026.08. 10.743912

In serial femtosecond crystallography (SFX), high sample consumption is a major hurdle to wider adoption, therefore method development has focused on lowering sample consumption. This is particularly important at high repetition X-ray free-electron laser sources such as the European XFEL, where fresh crystals must be delivered into the beam at MHz frequencies. Until now, high repetition rate sample delivery has mostly used fast thin liquid jets produced by gas-dynamic virtual nozzles. The majority of crystals in jets are delivered between XFEL pulses and are therefore wasted.
Fixed targets offer a sample delivery method with lower sample consumption, as crystals are immobilised on a surface so are not lost between pulses, but their use has so far been limited to lower repetition rates due to the limited speed of linear motion stages. Here we present a novel disc-shaped sample holder which spins at high speeds to enable high repetition rate, low sample consumption SFX at the SPB/SFX instrument of the European XFEL. This vacuum-compatible device sandwiches crystals embedded in viscous medium between two thin films on a 120 mm diameter disc, and has been used for successful data collection and structure solution.