Fluorescence-free First Hyperpolarizability Values of Fluorescent Proteins and Channel Rhodopsins

The fluorescence-free measurement of molecular second-order nonlinear optical properties (Le. the first hyperpolarizability beta) in solution for a set of fluorescent proteins and channel rhodopsins is reported. Because removing the fluorescence contribution is critical in providing accurate values for this nonlinear optical property, especially for the fluorescent proteins, we use and critically compare two different approaches to correct hyper Rayleigh scattering from the multiphoton fluorescence contribution. On the one hand, we use the frequency domain where the delayed fluorescence contribution is separated from the instantaneous hyper-Rayleigh scattering contribution through amplitude modulation and on the other hand we use the spectral domain where the broadband fluorescence spectrum is subtracted from the narrower hyper Rayleigh scattering line. The two methods, discussed in terms of their efficiency for routine measurements, yield similar first hyperpolarizability values, therefore establishing the robustness of the two approaches to correct for the fluorescence. From the data reported, the previously observed trend where more red-shifted fluorescent proteins have larger hyperpolarizability values because of their longer conjugated chromophore is unambiguously confirmed. For the channel rhodopsins, it is shown that despite their obligate dimeric and fairly symmetric nature, large first hyperpolarizabilities are measured, pointing to their potential use for second-harmonic imaging and membrane potential measurements in nonlinear optogenetics.