## 4 Data Collection Strategy for Pump-Probe TR-SFX Using a Highly Viscous Sample  
In the following section, the experimental protocol of pump-probe TR-SFX while using our optical-fiber-based setup with the OPO pump-laser system will be described, which might aid in efficiently conducting TR-SFX experiments. We first focused on the technical matters on pump illumination, such as the pump pulse energy, pump pulse repetition rate, pump focal size, and the sample flow rate, as the actual experimental design and conditions, particularly pump illumination conditions, may be challenging to establish. In crystallo TR spectroscopy should ideally determine the pump pulse energy at the sample point prior to TR-SFX experiments. In the TR-SFX study of bR, the pump energy was 4 \(\upmu\)J (2 \(\upmu\)J from each direction) with a \(\mathcal{O}\)40-\(\upmu\)m (FWHM) focal size, which was sufficient for the photoexcitation of the retinal in bR and it was below the threshold of sample damage by the pump pulse illumination. So far, the pump pulse energy used for TR-SFX was a few nJ/\(\upmu\)m\({}^{2}\) or less [7; 8; 9]. The pump pulse repetition rate is adjustable to be sub-multiple of 30 Hz, which is the XFEL repetition rate at SACLA. Normally, the pump pulse repetition rate is chosen to be 10 or 15 Hz. At a 15-Hz pump repetition rate, a diffraction pattern from a non-illuminated crystal (\"Dark\") is obtained at 33.3 ms after a diffraction pattern from a pump-illuminated crystal (\"Light\") is recorded. The \"Dark\" image is useful for the calculation of Fo(\"Light\") - Fo(\"Dark\") difference Fourier maps for capturing protein conformational changes. At a 10-Hz pump repetition rate, a \"Light\" image, followed by two \"Dark\" images (\"Dark1\" at 33.3 ms and \"Dark2\" at 66.6 ms after the pump illumination), are sequentially obtained. Here, it should be noted that the sample flow rate has to be fast enough to  
Figure 3: Time-resolved (TR) UV-visible spectra of bR in (**a**) a solution and (**b**) a microcrystal. exchange pump-illuminated crystals with non-illuminated crystals within 33.3 or 66.6 ms for the 15- or 10-Hz pump repetition conditions, respectively, to obtain the \"Dark\" images. A detail of the suitable sample flow rate will be described later. The pump focal size is adjustable from 20 to 250 \(\upmu\)m, while using different core size optical fibers. The best pump focal size depends on delay times (\(\Delta t\)) of interest. Basically, for TR-SFX measurements with \(\Delta t<\) -1 ms, the suitable pump focal size is about the diameter of the sample stream. However, for TR-SFX measurements with \(\Delta t>\) -1 ms, the pump focal size should be larger because the pump-illuminated crystals move significantly downstream during the period of \(\Delta t\). For example, the pump focal size was \(\mathcal{O}\)40 \(\upmu\)m in FWHM (\(\mathcal{O}\)90 \(\upmu\)m in full width) in the TR-SFX experiments for bR crystals with \(\Delta t<\) 1 ms (sample flow rate of 9.4 \(\upmu\)m/1 ms) [7], whereas the pump focal size was set to be \(\mathcal{O}\)250 \(\upmu\)m in TR-SFX experiments of P450 nor with \(\Delta t=\) 20 ms (sample flow rate of 4.6 \(\upmu\)m/1 ms) [9], where pump-illuminated crystals move approximately 90 \(\upmu\)m downstream within 20 ms. The large pump focal size of \(\mathcal{O}\)250 \(\upmu\)m allowed for crystals irradiation in the sample stream between the intersection point with XFEL and the position approximately 90-\(\upmu\)m upstream from it, which ensured the integrity of 'Light' data collection without being influenced by the sample flow fluctuation. The stability of the sample speed would be required so that a pump-illuminated crystal can interact with XFEL after a certain delay time if the pump focal area was set to be well upstream and distant from the intersection point with XFEL. In TR-SFX of P450 nor, the center of pump focal area was only aligned 45-\(\upmu\)m upstream from the XFEL beam (\(3\times 3\)\(\upmu\)m\({}^{2}\)), to secure the interaction of illuminated crystals with XFEL (Figure 4).