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See supplementary material at for: difference mass spectrum of [FL − H] (Fig. S1) and [FL + H]+ (Fig. S5); mass spectrum depicting the fragmentation enhancement effect required for tPF (Fig. S2); dependence of the one-color fragmentation efficiency of [FL − H] (Fig. S3) and [FL + H]+ (Fig. S6) on the intensity of the respective pump pulse; transient photofragmentation traces of [FL − H] recorded at parallel and perpendicular relative laser pulse polarization and at the magic angle (Fig. S4); ab initio calculated ground state geometries of [FL − H] and [FL + H]+ (Fig. S7) and the respective rotational constants/moments of inertia (Table SI); detailed description on the calculation of the orientational correlation functions of the second rank for freely rotating asymmetric top molecules.[Supplementary Material]
J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed. ( Springer, 2006), Chap. 10.

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Excited state dynamics of deprotonated and protonated fluorescein were investigated by polarization dependent femtosecond time-resolved pump-probe photofragmentation in a 3D ion trap. Transients of deprotonated fluorescein exhibit vibrational wavepacket dynamics with weak polarization dependence. Transients of protonated fluorescein show only effects of molecular alignment and rotational dephasing. The time resolved rotational anisotropy of protonated fluorescein is simulated by the calculated orientational correlation function. The observed differences between deprotonated and protonated fluorescein are ascribed to their different higher lying electronically excited states and corresponding structures. This is partially supported by time-dependent density functional theory calculations of the excited state structures.


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