Excited-State Proton Transfer Dynamics in LSSmOrange Studied by Time-Resolved Impulsive Stimulated Raman Spectroscopy.

LSSmOrange is a fluorescent protein that exhibits a large energy gap between absorption and emission, which makes it a useful tool for multicolor bioimaging. This characteristic of LSSmOrange originates from excited-state proton transfer (ESPT): The neutral chromophore is predominantly present in the ground state while the bright fluorescence is emitted from the anionic excited state after ESPT. Interestingly, it was reported that this ESPT process follows bimodal dynamics, but its origin has not clearly been understood. We investigate ESPT of LSSmOrange using time-resolved impulsive stimulated Raman spectroscopy (TR-ISRS) that provides femtosecond time-resolved Raman spectra. The results indicate that the bimodal ESPT dynamics originates from the structural heterogeneity of the chromophore. Species-associated Raman spectra obtained by spectral analysis based on singular value decomposition (SVD) suggest that