## 1 Structural Biology at XFEL Sources
### Structural Biology Experiments at XFEL Sources  
Traditional synchrotron-based crystallography experiments often use individual crystals mounted on goniometers to sample the rocking curve in order to measure a complete set of diffraction spot intensities [20]. Rotation of the crystal during each detector integration window (i.e. exposure) allows for measurement of the full scattering intensity of each Bragg reflection. Typically, a dataset comprises a few hundred detector images, each with an integration window of hundreds of milliseconds to seconds. Due to the high brilliance and X-ray pulse delivery patterns, macromolecular crystallography experiments at FEL sources typically employ a serial approach where each recorded diffraction image is produced from a new crystal, a method known as serial femtosecond crystallography (SFX) [21; 22]. Diffraction from each FEL pulse is recorded into a separate detector image, where the short pulse lengths'snap-shot' a crystal in a random orientation, recording only partial interactions of reciprocal lattice points and the Ewald sphere. Often, detector frames in excess of 10,000 are integrated to form a single complete dataset to account for the stochastic nature of the data collection [23]. FEL sources have the high intensity and short pulse durations necessary for high-resolution, time-resolved studies. Serial crystallography type experiments have also been successfully performed at synchrotron sources, often requiring concessions to beam intensity and/or bandwidth [24; 25]. The significantly large increase in peak brilliance of XFELs potentially provides crystallographers with an opportunity to complete otherwise impossible projects. Diffraction data from small nanocrystals typically require a brilliance not available at synchrotron sources. XFELs provide a feasible way to measure diffraction from crystals as small as 5 um [22] and in some cases even nanocrystals [26]. A number of variables contribute to the ultimate resolution of a crystallographic dataset, and there is a broad body of work that considers the intrinsic nature of crystal diffraction [27; 28; 29; 30]. Though beyond the scope of this review, broadly speaking, smaller crystals often yield poorer results than larger crystals from the same type of molecule. As the number of unit cells within the crystal increases by \(N\), the signal recorded is amplified by approximately \(N^{2}\). Producing protein crystals large enough for use in common crystallographic experiments can be a major hurdle for many projects. Significant time, cost, and optimization are needed to produce purified recombinant protein and, even with purified protein, unfortunately, crystallization processes do not always produce usable, diffracting crystals [31; 32; 33]. Serial femtosecond crystallography is a variant of crystallography that, as the name suggests, uses a serial approach to data collection in which crystals are injected into interaction region, typically via a liquid medium, and are then exposed to incident X-ray pulses resulting in a single crystal diffraction event [21; 34]. Notably, the liquid medium and surrounding gas used also scatters photons and causes an increase in the detected background signal. SFX injection techniques often optimize jet size to crystal size in order to minimize the background scatter and make post-experiment analysis easier. Best injection practices aim to maximize the stable jet length and delay the Rayleigh breakup point, where droplet formations occur, Figure 1[35; 36; 37; 38; 39]. Droplets formed have a diameter about 40% larger than the jet diameter and therefore contribute to more background signal. Furthermore, they are only present when the beam arrives some of the time, so without explicit timing, the proportion of pulses that intersect liquid containing crystal decreases. Additionally, the energy deposited into the liquid jet by an FEL X-ray pulse causes jet instability via rapid and violent vaporization, destroying the local stable region and the sample.