Polarization dependence of aligned collagen tissues imaged with second harmonic generation microscopy

A polarimetric second harmonic generation (SHG) microscope was used to analyze the dependence between polarization and SHG signal from collagen-based samples. A theoretical model was also developed to investigate the SHG intensity as a function of different polarization states for a set of quasiparallel fibers. Numerical simulations were compared to experimental SHG intensity values and a fairly good agreement was found. Linear polarized light produced periodical changes in the emitted SHG signal with a maximum of intensity corresponding to polarization parallel to the main orientation of the fibers, regardless the ratio of hyperpolarizabilities, ρρ . A similar behavior was found for elliptical states located along a vertical meridian on the Poincaré sphere (i.e., null azimuth) although the modulation of the SHG signal was different. Our numerical calculations described a dramatic change in this regular trend when ρρ changed from positive to negative values. Moreover, we provide an experimental method (based on the analysis of the modulation of the SHG signal) to determine the value of the ratio ρρ and, consequently, to obtain information about the internal organization of the collagen fibers.