Theoretical, numerical and experimental study of geometrical parameters that affect anisotropy measurements in polarization-resolved SHG microscopy

Polarization-resolved second harmonic generation (P-SHG) microscopy is an efficient imaging modality for in situ observation of biopolymers structure in tissues, providing information about their mean in-plane orientation and their molecular structure and 3D distribution. Nevertheless, P-SHG signal build-up in a strongly focused regime is not throroughly understood yet, preventing reliable and reproducible measurements. In this study, theoretical analysis, vectorial numerical simulations and experiments are performed to understand how geometrical parameters, such as excitation and collection numerical apertures and detection direction, affect P-SHG imaging in homogeneous collagen tissues. A good agreement is obtained in tendon and cornea, showing that detection geometry significantly affects the SHG anisotropy measurements, but not the measurements of collagen in-plane orientation.