Fluorescence-Free Spectral Dispersion of the Molecular First Hyperpolarizability of Bacteriorhodopsin

The molecular first hyperpolarizability (beta) of bacteriorhodopsin was measured over a broad spectral range (800-1300 nm) by means of a spectral hyper-Rayleigh scattering setup. We observed that two-photon fluorescence introduced a systematic error toward overestimating the beta value. Spectrally resolved hyper-Rayleigh scattering allowed us to quantify and correct for this effect, resulting in a drop to about one-half of the beta value reported in the literature. Moreover, we determined the dispersion of beta over the wavelength range between 800 and 1300 nm. Applications such as second harmonic generation (SHG) imaging rely on accurate molecular first hyperpolarizability values, along with information about resonance effects and wavelength-dependent photodamage. With new and more accurate measurement setups, it will be possible to gain more insight into the nonlinear optical response of bacteriorhodopsin.