Ratiometric indicators with long emission wavelength are highly preferred in modern imaging applications due to their elimination of influence from complex experimental conditions and large penetration depth. REX-GECO1 is a red fluorescent protein-based Ca²⁺ biosensor and was utilized in intensiometric and excitation ratiometric imaging with a long Stokes shift. To unravel its working mechanisms involving the photoexcited states, we implemented the steady-state electronic spectroscopy, ultrafast transient absorption (TA), quantum calculations, and molecular dynamics (MD) simulations. The pH-dependent absorption spectroscopy reveals the importance of a nearby Glu80 in determining the distinct electronic absorption profiles between the Ca²⁺-free and bound biosensors. Further 2D fluorescence map raises the possibility of implementing REX-GECO1 as the first green (510 nm)–red (610 nm) emission ratiometric biosensor with 480 nm excitation. Our femtosecond-TA data imply the trapping of the Ca²⁺-free protein chromophore in a dark excited state, characterized by an excited state absorption band centered at 532 nm which accumulates on the ~2 ps time scale. However, in the Ca²⁺-bound state, the biosensor chromophore undergoes excited-state proton transfer (ESPT) with a ~1.3 ps time constant as the dominant pathway. The nature of the dark state is likely a twisted conformation based on quantum calculations, further corroborated by MD simulation results. Moreover, tunable femtosecond stimulated Raman spectroscopy (FSRS) in the visible to near-IR region could help dissect the functional reaction coordinates in great detail. Furthermore, in vivo imaging in HeLa cells achieved a red/green emission ratio change (ΔR/R₀) of ~300%, which outperforms many FRET and single FP-based indicators. This comprehensive study offers new principles in designing the next-generation biosensors with larger dynamic range and longer emission wavelength.

Shuce Zhang

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