## 2 Pump-Probe Time-Resolved SFX Experiments
Furthermore, optical fibers facilitate the pump beams' alignment procedure, because the pump beam paths from the laser to fiber inputs and from fiber outputs to the objective lenses are normally fixed. Two-types of pump-laser systems are currently available: an optical parametric oscillator (OPO) providing \(>\)1 mJ, 6 ns pulses at a wavelength that is tunable from 300 to 2000 nm with a repetition rate of up to 30 Hz (NT230, EKSPLA) and an Nd:YAG laser that delivers 12 mJ, 5 ns pulses at 532 nm with a repetition rate up to 15 Hz (Minilite-I, Continuum). The focal size of the pump beams is changeable from 20 \(\upmu\)m (FWHM) to 250 \(\upmu\)m (top-hat), depending on fiber core size (50-400 \(\upmu\)m). The pump beam can be spatially aligned with the XFEL beam while using XYZ stages controllable from outside of the experimental hutch. The pump laser is synchronized with the XFEL via a pulse generator (DG645, Stanford Research Systems), which can be used to easily adjust the delay time between the pump and XFEL pulses. The timing jitter is \(<\)1 ns when the delay time is set to \(<\)16.7 ms. When considering the pulse duration (6 ns) of the OPO laser, the time resolution is \(<\)7 ns. It should be noted that the timing jitter is approximately 0.1 ms for the delay time longer than 16.7 ms.  
A helium gas flow path, where a focused XFEL beam passes through, is introduced between the beamline exit and the sample position to reduce background noise from air scattering. Limited spaces around the sample cannot afford the use of a helium chamber. A sample is ejected from an injector that was mounted on a motorized manipulator and it is delivered to an XFEL focal point. A high-viscosity sample injector for ejecting viscous samples [20; 21], such as crystals embedded in lipidic cubic phase (LCP) [22], is mainly used for the sample injection. Although a liquid jet injector, such as a gas dynamic virtual nozzle [23], is also applicable for sample delivery in pump-probe SFX, the available delay time is limited to within several microseconds, because the microcrystals that are illuminated by a pump laser flow down away from the intersection point with XFEL, basically due to its fast sample speed (approx.10 m/s). A high-viscous sample can be delivered with a slow speed (approx. 10 mm/s), stretching the maximum delay to several tenths of ms, to overcome this problem. However, there are concerns regarding the instability of a high-viscous sample flow, which might cause inaccuracy in the delay time or multiple sample illumination. In 2015, the developed setup for pump-probe TR-SFX was first demonstrated while using bR, which yields high quality microcrystals in LCP, with the Nd:YAG laser at a delay time (\(\Delta t\)) of 250 \(\upmu\)s after photoactivation to provide clear difference Fourier maps. After several experiments, the TR-SFX data were collected from the bR microcrystals after photoactivation for \(\Delta t\) = 16 ns, 40 ns, 110 ns, 290 ns,  
Figure 1: (**a**) Arrangement for pump-probe time-resolved serial femtosecond crystallography (TR-SFX) experiments using a nanosecond laser. An enlarged view around the sample interaction point is shown in (**b**). 760 ns, 2 us, 5.25 us, 13.8 us, 36.2 us, 95.2 us, 250 us, 657 us, and 1.725 ms at 2.1 angstrom resolution to provide 13 structural snapshots of conformational changes during proton transfer [7]. In the experiments, monoolein and paraffin oil were added into the bR microcrystals that were embedded in LCP before measurement to keep a stable flow from the highly viscous sample injector. In fact, the high-viscous mixture streams were recorded while using a high-speed camera to prove that the speed was stable (Figure 2). Moreover, [PERSON] et al. reported the viability of TR-SFX while using the LCP injector [20], with bR microcrystals in LCP at the beamline of the CXI at LCLS [24]. Other highly viscous carriers, such as grease [25] or hydroxyethyl cellulose [26], were also available for sample delivery in TR-SFX.