## 3 Time-Resolved Serial Femtosecond Crystallography
### TR-SFX with Light-Responsive Biomacromolecules
Such multiphoton excitation can lead to the propagation of radical intermediates, which obscures the biological interpretation of the TR-SFX data [63; 64]. Efficient photoexcitation protocol guided by the protein photochemical properties should be followed to allow for the biologically meaningful single-photon absorption of the biomolecules in crystals to avoid such an effect, i.e., multiphoton excitation. Moreover, examining the samples in their crystalline and solution forms with a static and time-resolved optical spectroscopy can significantly assist in the designing of the pump-probe TR-SFX experiments [28; 58; 61]. Such spectroscopic scanning, e.g., time-resolved UV-visible (transient) spectroscopy, can provide essential information that could help to establish the reaction photocycle, especially when examining a novel system [65], and estimate the time range of each of the photo-intermediates and highlight the differences, if any, between the crystalline and solution forms. As a result, the optimal photoexcitation wavelength and the duration of the photo-intermediates can be determined prior to the pump-probe TR-SFX experiment, thereby assisting in the  
Figure 2: Pump–probe MHz TR-SFX setup using the SPB/SFX instrument of the European XFEL. This setup was implemented during the very first pump–probe TR-SFX experiments [18]. In this setup, X-ray pulses arrive in 1.13 MHz bursts with each burst contains 176 X-ray pulses, which repeat every 100 ms. The fs-pump laser delivers 376 kHz pulses in the blue (\(\lambda\) = 420 nm), tunable based on the sample requirements, synchronized to the X-ray pulses which intersect precisely with the microcrystal jet produced by the GDVN. The pump–probe experimental scheme was designed as follows: one light triggered a (\(\Delta t\) = 0.89 \(\upmu\)s) pattern followed by two dark patterns (no light triggering: (\(\Delta t\) = 1.78 and 2.67 \(\upmu\)s)) to prevent light contamination from the previous laser excitation. The time delay between each pattern equaled 887 ns, corresponding to the X-ray pulse spacing at 1.13 MHz with each pattern representing a different probed crystal. The TR-SFX diffraction patterns were collected on an AGIPD detector (adapted from [18]). experimental design [61; 64; 66; 67]. [PERSON] et al. reported comprehensive details of the guidelines and parameters that need to be considered for the photo-excitation setup during pump-probe TR-SFX experiments [62]. As an example, to study the light-induced structural changes of photosystem (PSII) upon two flash (2F) excitation, [PERSON] and co-workers used an ATR-FTIR difference spectroscopy with similar pump laser setup to study the system both in solution and crystalline forms. This study allowed them to estimate the population of the S3 intermediate state upon 2F excitation in the microcrystalline form and used such information for their pump-probe TR-SFX experiment at SACLA [28]. On the other hand, it is also necessary to consider testing the sample delivery devices for their photon-resistance properties before using them for pump-probe TR-SFX experiments in cases when the illumination of the samples in the nozzle is required. This can be achieved by the visual inspection of the fabricated devices after illumination with a range of optical laser powers using a similar laser setup to that of the pump-probe TR-SFX approach [44; 45].