Femtosecond stimulated Raman spectroscopy (FSRS) is an ultrafast vibrational technique which allows rapid collection of Raman spectra with simultaneously high temporal and spectral resolution. With the recent development of FSRS methodology, three FSRS techniques (conventional, tunable, and anti-Stokes) have been implemented in our laboratory to dissect the excited state structural events of two photochemical systems: green fluorescent protein (GFP) based Ca2+-biosensors (macromolecules) and a photoacid pyranine called HPTS (small molecule). In my early work, I used FSRS to investigate the excited state proton transfer (ESPT) process in an intensiometric Ca2+-biosensor with green fluorescence, G-GECO1.1. The conventional FSRS effectively monitors the decay of the protonated chromophore species (A*) and reveals that a small-scale proton motion occurs on the sub-picosecond time scale upon photoexcitation and the ensuing ESPT process takes longer time in the Ca2+-bound biosensor. By strategically tuning the Raman pump in wavelength-tunable FSRS to track the deprotonated species (I*) in the excited state, the ESPT reaction time lengthening in the Ca2+-bound G-GECO1.1 is confirmed. Besides ESPT, an ultrafast chromophore twisting process and conformational inhomogeneity on the electronic excited state (ES) are also revealed. Later, conventional FSRS was used to dissect the fluorescence modulation mechanism of an excitation-ratiometric Ca2+-biosensor, GEX-GECO1. The distinct A* decay dynamics observed in the Ca2+-free and bound biosensors indicate that more structural inhomogeneity exists in the Ca2+-bound species, which is supported by the molecular dynamics (MD) simulation result. The lifetime of the remaining A* which does not transfer proton and the vibrational cooling (VC) process are also illuminated. Despite Ca2+-biosensors, the ESPT and VC process of a small photoacid, HPTS, are examined by our newly developed anti-Stokes FSRS. The VC process is revealed to exhibit a biphasic dynamic with an initial low-frequency motion aided energy dissipation process followed by thermal cooling to bulk solvents on longer time scale. Several interesting phenomena such as Raman gain and loss sign flip with the progress of reaction are observed, which enriches our understanding of this relatively new field. My future work includes: 1. Investigate the excited-state structural dynamics of an GFP S205V S65T variant and identify the effect of these mutations; 2. Elucidate the working mechanism of molecules with aggregation-induced emission (AIE) property; 3. Unravel the excited state process of a ferrous (II) porphyrazine complex in solution.
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