Nonlinear optical properties of mStrawberry and mCherry for second harmonic imaging

The second-order nonlinear optical properties of two monomeric red fluorescent proteins, mStrawberry and mCherry, have been experimentally determined by frequency-resolved femtosecond hyper-Rayleigh scattering. These proteins were found to exhibit a stronger nonlinear response than the previously described eGFP, eYFP and DsRed, confirming the trend that fluorophores with a more extended conjugated system, or a lower-energy band gap between ground and excited state, exhibit a larger static hyperpolarizability (β). Furthermore, these experimental data were complemented with quantum chemical calculations. A discrepancy was observed between experimental and theoretical results, but this could be explained by the chromophore model extracted from the available X-ray diffraction data. While eGFP showed a larger dynamic experimental response (β) due to the highest resonance enhancement, we measured an even higher signal for mCherry. Furthermore, mCherry also shows a better separation of the second harmonic signal and two-photon excited fluorescent signal, making this the preferred fluorescent protein for second harmonic imaging at 800 nm so far.