Structure sensitivity and sources of contrast in third-harmonic generation (THG) microscopy of cells and tissues

Abstract : Third-harmonic generation (THG) microscopy can provide structural information from unstained biological samples such as developing embryos. However, the contrast mechanisms in THG imaging need to be better characterized in order to develop practical applications. We studied experimentally and theoretically the influence of sample structure and excitation NA (Rayleigh length) on THG signals for various cases (spheres, interfaces). Because the third-harmonic signal critically depends on the spatial distribution of the Gouy shift, the effect of changing the excitation NA depends on the sample geometry within the focal volume. This phenomenon can be used to highlight certain structures within a complex system. Finally, we measured the nonlinear optical properties of several liquids, and we identified lipid bodies as an important source of contrast in biological THG imaging. We show that the technique can be used to characterize lipid accumulation in a variety of cells and tissues. © (2006) SPIE--The International Society for Optical Engineering