Polarization-resolved Second Harmonic microscopy in anisotropic thick tissues

We thoroughly analyze the linear propagation effects that affect polarization-resolved Second Harmonic Generation imaging of thick anisotropic tissues such as collagenous tissues. We develop a theoretical model that fully accounts for birefringence and diattenuation along the excitation propagation, and polarization scrambling upon scattering of the harmonic signal. We obtain an excellent agreement with polarizationresolved SHG images at increasing depth within a rat-tail tendon for both polarizations of the forward SHG signal. Most notably, we observe interference fringes due to birefringence in the SHG depth profile when excited at π/4 angle from the tendon axis. We also measure artifactual decrease of r = χ_xxx/χ_xyy with depth due to diattenuation of the excitation. We therefore derive a method that proves reliable to determine both r and the tendon birefringence and diattenuation.