Experimental and simulation study of the wavelength dependent second harmonic generation of collagen in scattering tissues.

We report on the wavelength dependence of second harmonic generation (SHG) of collagen in scattering tissues over the wavelength range of 800-1200 nm. The study incorporates inclusion of the molecular hyperpolarizability β of collagen and optical scattering, both of which are wavelength dependent. Using 3D SHG imaging and Monte Carlo simulations, we find the wavelength dependence of β is not well described by a two-state model based on known absorption bands. We further find that longer wavelength excitation is inefficient as the reduction in scattering is overcome by the decreased β far from resonance and the optimal excitation is within the 800-900 nm range. The impact is larger for backward collected SHG.