1. 1 Miyasaka Laboratory Yusuke Satoh David W. McCamant et al, Science, 2005, 310, 1006-1009 Structural observation of the primary isomerization in vision with femtosecond-stimulated Raman

2. 2 Vision The light reaches the retina through eyes and is changed into signal in retina. Signals are sent to our brains. Scheme 1. Structure of eye (Ref. http://www.kiriya-chem.co.jp/q&a/q52.html)

3. 3 Retinal Scheme 2. Structure of Rhodopsin Opsin is a protein of 7 spiral structures. A chromophore inside Opsin is Retinal. 11-Cis Retinal changes into all- trans-retinal by light irradiation. Signal is sent to the optic nerve. (Ref. http://www.spring8.or.jp/j/user_info/sp8-info/data/5-6-2k/5-6-2k-3-p394.pdf)

4. 4 Past research of retinal Table 1 Fluorescence lifetime and Transient absorption spectroscopy of retinal Ref. Chem. Phys. Lett., 2001, 334, 271 Science, 1991, 100, 14526 Transient absorption measurement and time-resolved fluorescence detection of 11-cis Retinal ～ 200 fs lifetime of the excited state reported.

5. 5 Motivation But fluorescence and electronic absorption spectra do not provide direct information of the molecular structure. A new time-resolved Raman spectroscopy method is necessary in order to elucidate the dynamics of this isomerization reaction and factors regulating this rapid structural change.

6. 6 Contents ・ Introduction ・ Experiment ・ Result and Discussion ・ Summary

7. 7 Principle of Spotaneous Raman scattering Stokes shiftAnti-stokes shift Virtual excited state Ground state Raman spectroscopy has been used for the identification of the chemical bond and for the determination of the molecular structure. Scheme 3. Mechanism of SpotaneousRaman scattering 0 ± ： Raman scattering  : Raman shift 0 0 － 0 ＋ 0

8. 8 Time-resolved Raman spectroscopy Pump pulse Sample Detector Intermed iate Scheme. 4 Time-resolved Raman spectroscopy The simple application of femtosecond laser pulse does not provide detailed information of vibrational spectra. Probe pulse Delay time 0 Raman scattering 0 －

9. 9 Resonance Raman and Stimulated Raman Excited state Ground state 0 0 － Resonance Raman Stimulated Raman Scheme. 5 Resonance Raman and Stimulated Raman Virtual excited state 0 0 － (narrow) + ( 0 －  (broad)

10. 10 Stimulated Raman spectroscopy Fig. 1. Stimulated Raman spectroscopy (Ref. Rev. Sci. Instrum., 2004, 75, 4971)

11. 11 Stimulated Raman system Fig. 2 Stimulated Raman spectroscopy system (Ref. Rev. Sci. Instrum., 2004, 75, 4971) Excited pulse: 500 nm, 30 fs fwhm Raman pump: 805 nm, 3 ps fwhm Raman probe: 830 ～ 960 nm, 20 fs fwhm

12. 12 Structures of 11-cis Retinal and all-trans Retinal 11-Cis Retinal change into all-trans Retinal by light irradiation. Fig. 3 Structure of 11-cis Retinal and all-trans Retinal

13. 13 Raman spectra of ground-state Retinal Fig. 4 Raman spectra of ground-state of 11-cis Retinal(bottom) and all-trans Retinal(top) ・ Raman spectra of 11-cis Retinal(bottom) 1548 cm -1 ・・・ C=C stretch 1100 ～ 1300 cm -1 ・・・ C-C single bond stretch and C-H rocking modes 969 cm -1 ・・・ hydrogen-out-of- plane(HOOP) wagging motion of the C 11 and C 12 hydrogens ・ Raman spectra of all-trans Retinal(top) 920, 875, and 850 cm -1 ・・・ C 11 -H, C 10 -H, and C 12 -H wagging mode hydrogen-out-of-plane(HOOP): 水素の面外変角運動 rocking mode: 横ゆれ変角運動 wagging mode: 縦ゆれ変角運動

14. 14 Time-resolved Raman spectra of Retinal Fig. 5 Time-resolved Raman spectra of Retinal(200 fs ～ 1 ps) and Raman spectra of ground state of 11-cis retinal(bottom) and all-trans retinal(top) The dispersive HOOP features evolve on the same time scale as the finger-print bands into the expected three positive features of the Bathorhodopsin spectrum. These data show that there is considerable reactive evolution on the ground-state surface from 200 fs to 1 ps.

15. 15 Time Profile of C 10 -H,C 11 -H and C 12 -H hydrogen wagging frequencies Fig. 6 Time profile of C 10 -H, C 11 -H and C 12 -H hydrogen wagging frequency The HOOP frequency increase by 100 cm -1 with 325 fs time constant.

16. 16 Fig. 7 Retinal chromophore structures for reactant rhodopsin and for photorhodopsin and bathorhodopsin that reproduce the observed hydrogen wagging frequencies. Structures of Retinal, Photorhodopsin and Bathorhodopsin The Bathorhodopsin structure is twisted by –144° about the C 11 =C 12 and by 31°about the C 12 –C 13 bond. The Photorhodopsin structure is more highly distorted, in particular about the C 9 =C 10 (45°), C 10 –C 11 (25°), and C 11 =C 12 (–110°) bonds. With these larger twists, the overall shape of retinal is much more like that of 11-cis Rhodopsin than all-trans Bathorhodopsin,

17. 17 Theoretical and experimental hydrogen wagging frequencies Fig. 8 Theoretical and experimental hydrogen wagging frequencies for the Photo and Bathorhodopsin structures Caluculated frequency for Photorhodopsin structure show good agreement with experimental data for the C 10 -H,C 11 -H modes. Vibrational calculations for the Bathorhodopsin structure yielded features in excellent agreement with experimental data, except for an underestimated C 11 –H wagging frequency.

18. 18 The isomerization coordinate for the primary event in vision Fig. 9 Multidimensional representation of the isomerization coordinate for the primary event in vision Excited-state of 11-cis Retinal carry the system toward a conical intersection in ～ 50 fs. From 200 fs to 1 ps, Photorhdopsin changes into Bathorhodopsin on the ground-state surface.

19. 19 Summary ・ Excited-state decay (200 fs) through a conical intersection is mediated largely by fast HOOP motion. ・ By 1 ps, vibrational cooling has narrowed, thereby completing the transformation to Bathorhodopsin.

20. 20 Stimulated Raman spectroscopy Fig. 1 Mechanism of stimulated Raman spectroscopy Amplitude of coherent vibration induced by Raman and probe pulse Heterodyne detection yields a gain feature on top of the probe envelope in the energy domain shifted in energy relative to the Raman pulse according to the frequency of the vibration. ① ① Stimulated Raman spectroscopy is obtained by this method.

21. 21 Retinal Scheme 2. Structure of Rhodopsin Opsin is a protein with 7 spiral structures. A chromophore inside Opsin is Retinal. 11-cis retinal changes into all- trans-retinal by light irradiation. Signal is sent to the optic nerve. (Ref. http://www.kiriya-chem.co.jp/q&a/q52.html)

22. 22 Feynman diagram

23. 23 Photoisomerization reaction of Rhodopsin

24. 24 Principle of Raman scattering Scheme 3. Mechanism of Raman scattering 0 ± ： Raman scattering  : Raman shift Raman spectroscopy has been used for the identification of the chemical bond and for the determination of the molecular structure. (Ref. http://www.natc.co.jp/bunseki/lr.html)

25. 25 Wagging mode and Rocking mode