## 3 Time-Resolved Serial Femtosecond Crystallography
### TR-SFX with Light-Responsive Biomacromolecules  
In the last decade and with the advent of hard X-ray free electron laser technology, the pump-probe TR-SFX approach has witnessed a significant development that has led to a spectacular advancement in the structural and dynamical studies of some highly challenging biological systems, i.e., light-responsive biological systems, such as photosynthetic proteins, phytochromes and microbial rhodopsins [24; 25; 28; 31; 55; 56; 57]. In 2012, Aquila et al. conducted the first TR-SFX experiments using the CFEL-ASG multi-purpose instrument at the Linac Coherent Light Source (LCLS) on the photosystem I-ferredoxin complex, where they used an Nd:YLF optical pump laser system to illuminate the complex and then probed the photo-induced structural changes that occurred within 5 to 10 \(\upmu\)s delay times using the X-ray free electron laser [23]. This pioneering pump-probe TR-SFX experiment, accompanied by the advances in the application of pump lasers and microcrystal delivery methods, has paved the way to rapid development within the study of the structure and dynamics of light-sensitive biomacromolecules [18; 24; 26; 27; 58; 28]. In the pump-probe TR-SFX, an optical 'pump' laser is used to trigger the biochemical reaction _in crystallo_ by illuminating the microcrystals with defined optical laser pulses at a defined wavelength and energy in a controlled region of the jet and/or nozzle before probing the reactions _in crystallo_ with X-ray FEL pulses 'probe' at different delay times to allow observing the reaction of the biomacromolecule in real time (Figure 2) [18; 23; 24]. There are several parameters that need to be considered and carefully tuned for a successful pump-probe TR-SFX experiment. The most obvious are the timing synchronization between the optical laser \"pump\" and the FEL \"probe\", the optical laser power, the materials used for the fabrication of the injection device and the optical density or the thickness and size of the microcrystals [18; 23; 54; 59; 60; 61; 62]. The synchronization of the X-ray FEL and optical laser pulses is critical for the accurate measurement of delay time points for experiments targeting the dynamics of timescales from fs to a few ps and hence, the data interpretation of the pump-probe SFX experiments, particularly for the instruments that run at a megahertz repetition rate [11; 54; 60]. With the SPB/SFX instrument at the European XFEL, significant progress has been made by improving the temporal resolution and decreasing the shot-to-shot timing fluctuations to the lowest ever megahertz rate timing jitter of only 24.0 fs RMS, with an uncertainty of 12.4 fs [54] and the ongoing development to further reduce the jitter. In 2020, [PERSON] et al. used micron-sized photoactive yellow protein (PYP) crystals to explore the initial isomerization events of the \(p\)-coumarin chromophore as well as the early stage intermediates of the photocycle, as early as 10-100 psdelay time points using the SPB/SFX instrument operating at MHz repetition rate [18]. The TR-SFX datasets showed excellent difference electron densities and allowed the authors to observe the clear torsional angle increase in the \(p\)-coumarin skeleton along the 10 to 30 ps before it relaxed at 80 to 100 ps delay time points, which corroborated well with shorter and longer time points collected at LCLS and APS, respectively [18; 26]. This early study of PYP using the SPB/SFX instrument demonstrated the feasibility of the pump-probe TR-SFX experiments at high repetition rate X-ray lasers and paved the way toward further improvements in the femtosecond temporal resolution (reviewed in [13]). The selection of the optical pump setup, e.g., the laser wavelength, fluence and pulse duration, requires careful experimental planning to achieve as high as possible excited state population while avoiding contamination, which may occur, for instance, due to the multiphoton excitation of the chromophores [15; 58; 61; 63].