Quantitative characterization of biological liquids for third-harmonic generation microscopy

Third-harmonic generation (THG) microscopy provides images of unstained biological samples based on spatial variations in third-order nonlinear susceptibility, refractive index, and dispersion. In this study, we establish quantitative values for the third-order nonlinear susceptibilities of several solvents (water, ethanol, glycerol), physiological aqueous (ions, amino acids, polypeptides, bovine serum albumin, glucose) and lipid (triglycerides, cholesterol) solutions as a function of solute concentration in the 1.05-1.25 μm excitation range. We use these data in conjunction with imaging experiments to show that THG imaging with ∼1.2 μm excitation lacks specificity and sensitivity to detect physiological ion concentration changes, and that nonaqueous structures such as lipid bodies provide a more robust source of signal. Finally, we illustrate the impact of index-matching liquids in THG images. These data provide a basis for interpreting biological THG images and for developing additional applications.