We report on third-harmonic generation (THG) of biomolecular solutions at the fluid/glass interface as a means to probe resonant contributions to their nonlinear absorption spectra that could serve as contrast mechanisms for functional imaging. Our source was 100 fs laser pulses whose center wavelength varied from 760 to 1000 nm. We find evidence of a two-photon resonance in the ratio of third-order susceptibilities, χ(3)sample (3ω)/χ(3)glass, for aqueous solutions of Rhodamine B, Fura-2, and hemoglobin and a three-photon resonance in χ(3)sample (3ω)/χ(3)glass for solutions of bovine serum albumin. Consistent with past work, we find evidence of a one-photon resonance of χ(3)sample (3ω)/χ(3)glass for water, while confirming a lack of resonant enhancement for benzene. At physiological concentrations, hemoglobin in different ligand-binding states could be distinguished on the basis of features of its THG spectrum.
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Clay et al. (2006) studied this question.