Second-order nonlinear optical properties of fluorescent proteins for second-harmonic imaging

The second-order nonlinear optical properties of three fluorescent proteins (FPs) (green, EGFP; yellow, EYFP; and red, DsRed) have been experimentally determined by frequency-resolved femtosecond hyper-Rayleigh scattering. As expected, DsRed, with its lower-energy bandgap between ground and excited state, exhibits the largest intrinsic hyperpolarizability. The anomalously low first hyperpolarizability for the yellow variant has been rationalized in terms of the centrosymmetrical arrangement between the phenolic Tyr203 (Tyr = tyrosine) residue and the chromophoric Tyr66 moiety, leaving the small imidazolinone moiety as the only effective non-centrosymmetric chromophore for second-order nonlinear effects. The experimental findings are corroborated by high-level computational results and suggest molecular engineering strategies to produce a full rainbow of FPs with enhanced nonlinear optical properties.