## 2 Pump-Probe Time-Resolved SFX Experiments  
The pump-probe technique for TR-SFX has been well established for the past five years. Here, optical pulses (\"pump\") illuminate a continuous flow of protein microcrystals, followed by the XFEL pulses (\"probe\") illuminating with various delay times to obtain time-resolved (TR) diffraction images. The important applications of this technique include direct observations of structural changes in photosensitive proteins. In general, relatively large movements in a protein, such as helix movements, occur in microsecond- and millisecond-timescales, while local motions in a protein, such as photoisomerization of a chromophore, proceed on the ultrashort timescale of femtoseconds to picoseconds [10]. The first experiment of time-resolved SFX was conducted in 2012 while using an optical pump laser that was synchronized with an XFEL of the LCLS to obtain X-ray diffraction images from the photoactivated state of a large membrane-protein complex [11]. In this experiment, photosystem I-ferredoxin co-crystals that were flowing in a liquid jet were irradiated with the optical pump laser and then shot by the XFEL, which resulted in light-induced changes at time delays of 5 to 10 \(\upmu\)s. The combination of the liquid jet sample injector and an optical pump laser was also used for the early time-resolved SFX studies (photoactive yellow protein [12; 13], myoglobin [14]). For instance, the trans-to-cis isomerization of the chromophore in photoactive yellow protein (PYP) was observed to arise at approximately a 590-fs time delay after photoexcitation [13], and the displacement of side chains in the chromophore pocket in PYP was observed to occur at a 1-\(\upmu\)s time delay [12]. These studies were performed at the Coherent X-ray Imaging (CXI) beamline of the LCLS [15], where the sample was delivered to an X-ray interaction point by a liquid jet injector in a vacuum chamber that was installed at the CXI. Data collection in a vacuum has the advantage of low background noise, as well as the avoidance of X-ray attenuation; however, evaporative cooling might cause temperature changes on a sample stream. Moreover, a vacuum environment offers less flexibility when installing devices, such as optics for a pump laser or a sample injector. Meanwhile, a helium chamber was introduced for the SFX experiments at the beginning of user operation at SACLA [16]. Sample delivery in SFX requires a thin stream with a high density of microcrystals to ensure a high hit rate; however, it is prone to clogging the nozzle of an injector. A helium atmosphere environment allows for the easy replacement of an injector and provides more flexibility in setting devices when compared to a vacuum environment, thus facilitating SFX data collection [17]. This experimental setup (Diverse Application Platform for Hard X-ray diffractioN In SACLA (DAPHNIS)), which consists of a helium chamber, a detector, an injector manipulator, and microscopes for the alignment of sample position, has been used for SFX experiments since 2013 [16]. In 2015, a new design of an experimental setup for pump-probe TR-SFX was adopted for achieving a high photoexcitation efficiency of samples and facilitating data collection. Figure 1 shows the recent setup for TR-SFX [18], which is composed of a pump-laser system, an injector manipulator, microscopes, and a multi-port readout charge-coupled device detector [19]. A nanosecond pump laser is incorporated into the setup to capture relatively large movements in proteins that occur at a delay time that is slower than nanoseconds. A pump-laser beam is divided into two arms while using a 50:50 beamsplitter, and each branch is fed into an optical fiber and then delivered near a sample stream. Each output beam from an optical fiber is collimated while using a fiber-coupled collimator and focused on a sample stream using an objective lens. The double-beam delivery system enables the illumination of protein crystals from two directions to enhance the excitation efficiency.