## 3 Time-Resolved Serial Femtosecond Crystallography
### TR-SFX with Light-Unresponsive Biomacromolecules  
TR-SFX requires a triggering method that is faster than the reaction mechanism steps, the homogenous triggering of the sample, i.e., each single crystal, and a high triggering efficiency. The \"low-hanging fruit\" of protein targets for TR-SFX studies are photo-reactive proteins, which account for only 0.5% of all proteins, whereas the majority are light-unresponsive proteins [68; 69]. As research interest moves away from just photoactive proteins, demand for alternative triggering methods increases. However, if the research target is simultaneously light-unresponsive and mechanistically fast, methods such as rapid mixing (ms) [32; 70; 71] and even temperature jumps (ns) [68] become too slow, with light triggering methods remaining at the fastest timescales (up to fs bond breaking timescale [72]). A promising method to artificially implement light sensitivity is the use of photocages. At a first glance, it may seem that the available compounds and research of new generic photocages will quickly fulfill the need for structural biology compatible photocages. However, the requirements for these experiments become restrictive to many available photocages. The solubility, cleavage rate, light absorption profile and extinction coefficient are all intertwined and need to collectively satisfy the requirements of the experiment [36]. Research has been conducted to find or manufacture new photocages, but even these have their limitations and the demand for new photocages still persists. There are various photocage factors to consider when planning TR-SFX experiments. The cleavage rate of a photocage relates to the speed from photon absorption to cage release, which is required to be no slower than the phenomenon the experiment is trying to observe, as the slowest event will restrain experiments' time resolution [72]. Photocage light absorption profiles, including both the absorption spectrum and the extinction coefficient, need to be compatible with the sample and the setup. If possible, the pump laser wavelength should not be close to the sample's absorption profile, i.e., proteins 280 nm or buffer, to minimize laser absorption by molecules other than those of the photocage [36]. The higher the extinction coefficient at a given wavelength, the less laser energy is required for efficient photocage light absorbance and hence, the less unwanted sample absorption, and vice-versa for lower coefficients. However, if the extinction coefficient is too high, problems can also arise with triggering homogeneity due to low transmission values along the laser path, as most light will be absorbed at the external crystal layers or in its surrounding buffer [72]. The quantum yield is the fraction of photocages that will release the caged compounds per photon absorbed. If the yield is low, higher pump energy or photocage concentration is required, but then again, these values will need to be compatible with the absorption profile. Other factors are solubility, compound stability and availability, i.e., either commercially available or manufacturable. Considering all previously mentioned factors and experimental requirements, it becomes obvious that a perfect photocage for each experiment is unlikely; however, a compromise between the various variables is achievable. Photocage-protein interaction is also relevant [36]. Regarding the photocage position within the crystals, there are two main possibilities: either the photocage interacts with a specific portion of the protein (i.e., active site) or it does not and is instead found dissolved along the solvent crystal channels. If the photocage specifically interacts with the active site, a high quantum yield will have increased relevancy as each interacting caged compound will only have one chance to trigger the reaction. Each active site where the caged compound remains bound due to low quantum yields will directly contribute to reaction triggering heterogeneity.