###### Abstract  
This perspective review describes emerging techniques and future opportunities for time-resolved serial femtosecond crystallography (TR-SFX) experiments using high repetition rate XFEL sources. High repetition rate sources are becoming more available with the European XFEL in operation and the recently upgraded LCLS-II will be available in the near future. One efficient use of these facilities for TR-SFX relies on pump-probe experiments using a laser to trigger a reaction of light-responsive proteins or mix-and-inject experiments for light-unresponsive proteins. With the view to widen the application of TR-SFX, the promising field of photocaged compounds is under development, which allows the very fast laser triggering of reactions that is no longer limited to naturally light-responsive samples. In addition to reaction triggering, a key concern when performing an SFX experiment is efficient sample usage, which is a main focus of new high repetition rate-compatible sample delivery methods. megahertz repetition rate; XFEL; SPB/SFX scientific instrument; serial femtosecond crystallography; time-resolved serial femtosecond crystallography; sample delivery; liquid jet; pump-probe; photocag +
Footnote †: journal:  
_Applied_  
Footnote †: journal:  
interaction point, in all but exceptional circumstances, only one diffraction pattern is ever collected per crystal, meaning that a dataset needs the exposure of and data collection from several thousands to hundreds of thousands of crystals to be completed [3]. This number of required samples is not trivial to deliver, nor is such a large amount of data necessarily easy to collect. The European X-ray Free Electron Laser (EuXFEL) in Schenefeld, Germany, is the only running source of megahertz repetition rate XFEL pulses currently available to users. With the Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of the EuXFEL that is equipped for SFX experiments, the typical X-ray pattern consists of X-ray pulse trains delivered at 10 Hz with each train comprising up to 352 pulses separated by 880 ns, leading to an intra-train repetition rate of 1.1 MHz [10; 11]. In its current configuration, a 1 Megapixel Adaptive Gain Integrating Pixel Detector (AGIPD 1M) is able to collect data at megahertz repetition rate, but it only operates in a vacuum environment [11; 12]. See [13] and references therein for a detailed description of a number of SFX experiments performed using the SPB/SFX instrument. The most important advantage of SFX at high repetition rate facilities is the ability to collect data at a much faster rate than other XFELs: at least 30 times faster for the EuXFEL [5; 9; 14; 15]. In practice, this means that 10 min of data collection at the SPB/SFX instrument produces the same amount of data as more than 5 h of data collection at any other currently available XFEL running at a low repetition rate. However, megahertz repetition rate and sample injection in vacuum come with limitations that need to be overcome, such as the sample needing to be replaced at the same rate and without freezing. Due to these constraints, the most commonly used sample delivery method is within a low viscosity liquid jet [16; 17]. Currently, it is the only method available to replenish the sample fast enough to bring a fresh crystal to the interaction point before the subsequent X-ray pulse is delivered while still remaining flexible enough to allow time-resolved studies in the form of mix-and-inject and pump-probe experiments [18; 19; 20]. Alternative methods are being tested as to whether they can achieve better results than the standard liquid jets while remaining compatible with the previously mentioned constraints. Time-resolved serial femtosecond crystallography (TR-SFX) is defined by the study of biomacromolecular reactions observed with time resolutions as rapid as femtosecond-scale using SFX [15; 21; 22].