Photochemistry powers numerous processes from luminescence and human vision, to light harvesting. However, the elucidation of multidimensional photochemical reaction coordinates on molecular timescales remains challenging. We developed wavelength-tunable femtosecond stimulated Raman spectroscopy to simultaneously achieve pre-resonance enhancement for transient reactant and product species of the widely used photoacid pyranine undergoing excited-state proton transfer (ESPT) reaction in solution. In the low-frequency region, the 280 cm⁻¹ ring deformation mode following 400 nm photoexcitation exhibits pronounced intensity oscillations on the sub-picosecond timescale due to anharmonic vibrational coupling to the 180 cm⁻¹ hydrogen-bond stretching mode only in ESPT-capable solvents, indicating a primary event of functional relevance. This leads to the contact ion pair formation on the 3 ps timescale before diffusion-controlled separation. The intermolecular 180 cm⁻¹ mode also reveals vibrational cooling time constants, ∼500 fs and 45 ps in both H₂O and D₂O, which differ from ESPT time constants of ∼3/8 and 90/250 ps in H₂O/D₂O, respectively. Spectral results using H₂ ¹⁸O further substantiate the functional role of the intermolecular 180 cm⁻¹ mode in modulating the distance between proton donor and acceptor and forming the transient ion pair. The direct observation of molecular structural evolution across a wide spectral region during photochemical reactions enriches our fundamental understanding of potential energy surface and holds the key to advancing energy and biological sciences with exceptional atomic and temporal precision.

Significance: Fluorescent proteins (FPs) started their incredible, colorful journey in bioimaging and biomedicine with the extraction and purification of GFP from the Pacific jellyfish Aequorea victoria more than 50 years ago. Recently, an expanded palette of genetically encodable Ca ²⁺ -sensing FPs have paved the way to image neural activities and important biological processes where Ca ²⁺ is the ubiquitous messenger. To unravel the molecular choreography of FPs engineered for visualizing Ca ²⁺ movement, we study the embedded chromophore upon photoexcitation and monitor its subsequent excited-state structural evolution with femtosecond Raman spectroscopy. The vivid insights on H-bonding network and functional roles played by strategic mutations provide a deep understanding of excited-state processes in biology and will guide future bioengineering efforts toward better biosensors.