Abstract
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 ρ = Χxxx/Χxyy with depth due to diattenuation of the excitation. We therefore derive a method that proves reliable to determine both ρ and the tendon birefringence and diattenuation.
Highlights
Second Harmonic Generation (SHG) microscopy is an efficient imaging technique to visualize the three-dimensional (3D) distribution of fibrillar collagen in biological tissues [1, 2]
Transverse profiles show that the SHG signal is higher at the tendon edges as expected because of the cylindrical shape of the tendon: attenuation of the excitation beam is more effective in the center of the section when the incident light propagates along a larger distance within the tendon
Way, depending on the propagation distance within the tendon. It results in interference fringes in the depth profile of the SHG signal for incident excitation mixing up x and y-polarization components
Summary
Second Harmonic Generation (SHG) microscopy is an efficient imaging technique to visualize the three-dimensional (3D) distribution of fibrillar collagen in biological tissues [1, 2]. The usual approach is to measure the ratio ρ of the two main tensorial components of the nonlinear response, considering a cylindrical symmetry for fibrillar collagen [9, 10, 11, 12, 13, 14]. This ratio depends on the orientational distribution of the collagen triple helices and on the orientation of the nonlinear dipoles (along the peptidic bonds) within the triple helix. Comparison of healthy and pathological tissue requires to characterize the accuracy of ρ measure-
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