## 4 Data Collection Strategy for Pump-Probe TR-SFX Using a Highly Viscous Sample
The sample flow rate in pump-probe TR-SFX should be sufficiently fast for the \"Dark\" data collection to exchange the photoexcited crystals to the dark state crystals within 33.3 or 66.6 ms, while a sufficiently slow rate is needed for the \"Light\" data collection to ensure the interaction of the photoexcited crystals with an XFEL pulse. In the TR-SFX of bR experiments, the microcrystals in LCP were extruded at a flow rate of 2.5 \(\upmu\)L/min. (9.4 \(\upmu\)m/1 ms) from a nozzle of 75-\(\upmu\)m internal diameter under the pump energy and size conditions that are mentioned above. Empirically, a sample flow rate of 5-10 \(\upmu\)m/1 ms might be a good first choice for the design of more detailed experimental conditions. It should be noted that the pump-light scattering from the sample stream could pre-excite the sample being upstream. Therefore, care should be taken on the \"Dark\" data collection. A sample flow rate of \(>\)10 \(\upmu\)m/1 ms might be needed, depending on the pump pulse energy and focal size. A 10-Hz pump repetition condition is a better choice than a 15-Hz condition in terms of the \"Dark\" data collection. The 10-Hz condition provides a sample exchange time of 66.6 ms, which is twice as long as the 15 Hz condition (33.3 ms). Thus, the sample flow rate can be reduced for the 10-Hz condition, which is twice as slow as for the 15-Hz condition. With the above technical points in mind, in the XFEL beamtime, we performed the pump-probe TR-SFX according to the following protocol:  
1. The data processing pipeline [33], which was based on Cheetah [34], was used for real-time feedback during the beamtime, hit finding, and sorting of \"Light\" and \"Dark\" images. In the pump-laser setup, a portion of the pump beam (-5%) was picked off and its intensity signal that was detected by a photodiode was used to tag the image as \"Light\". Figure 4: Spatial alignment of the pump-illuminated area. Protein microcrystals that were embedded in the carrier media were injected to the X-ray beam interaction point while using the high-viscosity sample injector [21]. The stability of the sample streams was essential for pump-probe TR-SFX experiments, because the pump-illuminated crystals must be exchanged with the fresh ones before the \"Dark\" data collection. 3. The injector height was aligned by using the sample stage (Z direction), so that the injector bottom was distant to the XFEL focal point by 300 \(\upmu\)m. The monitor image of Microscope 1 was helpful (Figure 1) for the alignment. 4. The sample stream was aligned to the XFEL focal point by using the sample stage (X direction). The monitor image of Microscope 1 was also helpful for this alignment. The hit rate was maximized in the best alignment. 5. The sample stream was aligned to the pump focal area by using the sample stage (Y direction). The monitor image of Microscope 2 was helpful in this alignment (Figure 1). In the best alignment, the pump-light scattering from the sample stream was maximized. 6. The delay time (\(\Delta t\)) between the pump and XFEL pulses, which is controlled by a pulse generator (DG645, Stanford Research Systems), was set. The timing jitter for \(\Delta t<\) 16.7 ms was \(<\)1 ns, but it was approximately 0.1 ms for \(\Delta t>\) 16.7 ms at SACLA. 7. Diffraction images were collected: typically, the hit rate of highly viscous samples is 20-30% by adjusting the crystal density to avoid multiple hits. 10,000-15,000 diffraction patterns are collected as hit \"Light\" images in an hour in the case of the 15-Hz pump laser condition. Given the typical indexing rate of 60-70%, 6000-10,000 indexed images are obtained in an hour. Because \(\sim\)20,000 indexed images are needed to clearly visualize the differences between \"Light\" and \"Dark\", one dataset takes 3-4 h, including the time for sample replenishment. 8. Measurement was repeated by using different values of \(\Delta t\) to afford a molecular movie.