## 2 Sample Delivery Methods
### Innovative Sample Delivery Methods  
Efficient sample consumption is one of the key elements of emerging and innovative injection delivery systems. The methods that allow for a lower sample consumption, such as tape drive, some drop-on-demand injectors [47; 48] and fixed target or viscous media injecting devices [49; 50], are incompatible or very difficult to adapt to megahertz rate operation in a vacuum. Fixed target holders that are compatible with operation at high  
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]. repetition rates are possible and are currently under development, but they are still far from ready and available as standard. 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. Even without the synchronization of the droplet with the X-ray pulse train, this approach has already shown a sample reduction of 60%. Theoretically, with perfect synchronization, sample consumption could be reduced by 99%, as no sample would be injected within the period where X-rays are not delivered (so called \"dark\" or \"dead\" time). Recent work at the EuXFEL has demonstrated the use of a variation of the segmented flow method in the form of a hybrid microfluidic 3D-printed co-flow device coupled with a GDVN [44] (Figure 1d). By injecting the sample immediately before injection through a side channel into the main capillary, which contains an immiscible oil, and tuning the flow rates, they have managed to deliver a stable liquid jet and reduce clogging effects. With this method, sample consumption could theoretically be reduced by 95%, whereas in this study sample, consumption was reduced by 75%. This reduction is achieved by lowering the sample flow rate that is needed to establish the liquid jet, but sample delivery is still continuous and thus, wasted during the \"dark\" time. The optical bunching method (Figure 1e) relies on the use of a laser setup to create an optical trap in the capillary to periodically trap and release concentrated crystals [46]. In the first description of this method, [PERSON] et al. demonstrated a 30-fold local sample increase in the concentration into 10 Hz bunches using polystyrene particles. If this technique is proven to be usable with biomacromolecular crystals during sample delivery with a GDVN, it could theoretically allow for a 20-fold reduction in sample concentration without impacting the hit rate compared to the regular liquid jets with a GDVN and thus, lead to a 95% reduction in the sample consumption. The development of these innovative sample delivery methods focuses on preserving the advantages of liquid jets, i.e., being able to deliver samples fast enough for the high repetition rate XFELs without freezing the sample in a vacuum as well as its flexibility, while addressing its major limitation of the sample wastage. This development raises other questions and difficulties.