## 3 In Crystallo TR Ultraviolet-Visible Spectroscopy  
The rate-limiting steps of protein reactions and, thus, the rates of intermediate formation and decomposition, generally depend on experimental conditions, such as temperature, pH, pressure, and solvent viscosity, among others. As such, the kinetics and detectable reaction intermediates in the crystalline phase are not necessarily identical to those in the solution phase. In this case, TR spectroscopy is useful in the evaluation of the in crystallo reaction kinetics. Thus, we performed TR visible absorption spectroscopy for bR microcrystals in the study of bR, which was key to the success of pump-probe TR-SFX.  
Details of the visible microspectrometer were described previously [7; 9]. In brief, the nanosecond, 532-nm pulse from an Nd:YAG laser (Minilite-I, Continuum) and the microsecond, white light pulse from a Xe-flash lamp were used as the pump and probe pulses, and they were both focused on the sample with \(\O{300}\) and \(\O{0100}\)\(\upmu\)m, respectively. The slurries of protein microcrystals were packed with a carrier medium (LCP in the case of bR) between two quartz windows with a 100-\(\upmu\)m spacer and were then placed at the probe focal point. It should be noted that the microcrystal size of bR (typically, 20 \(\upmu\)m) was smaller than the probe focal size, so that microcrystal ensembles, containing multiple microcrystals with random orientations, were measured. The sample cell temperature was kept at 20 \({}^{\circ}\)C with a water-cooled chiller.  
Figure 3 shows the microsecond-TR visible absorption spectra of bR in the solution and the microcrystal upon photoexcitation at 532 nm. The figure illustrated the difference spectra, which were constructed by subtracting the dark state (bR570) spectrum from each TR spectrum. Here, the negative difference signal arose from bR570. The TR spectra of bR solution showed K590, L550, M412, and O640 intermediates in the photocycle, as previously reported [29]. The N560 intermediate did not significantly accumulate under the condition used (pH 5.5) [30]. The TR spectra of bR microcrystal showed no red-shifted peaks corresponding to K590 and O640. The global fitting analysis revealed that, in the microcrystal, L550 appeared within the current time resolution (\(<\)1 \(\upmu\)s) and it was converted to M412 with a half-life time of 15 \(\upmu\)s [7]; bR570 was then recovered with a half-life time of 8 ms, without the accumulation of O640, probably because the crystal packing affected the rate-limiting step of the reaction. However, it is known that an initial proton pump step after the retinal photoexcitation occurred during the transition of L550 to M412, accompanied by deprotonation of the retinal. Therefore, L550 and M412, as well as early K590 (\(<\)1 \(\upmu\)s), were focused on in the TR-SFX experiment based on the TR spectroscopic results. These spectroscopic results on bR microcrystals underline the fact that TR-SFX should be used in combination with in crystallo TR spectroscopy. So far, in crystallo TR X-ray emission spectroscopy [31] and in crystallo TR-IR spectroscopy [32] have also been successfully combined to TR-SFX. Such combinatorial use of various TR spectroscopies, including Raman scattering, fluorescence, electron spin resonance, and so on, should be increasingly applied.