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 polarization-resolved 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 pi/4 angle from the tendon axis. We also measure artifactual decrease of rho = chi(xxx)/chi(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. (C) 2010 Optical Society of America