Digital polarimetric second harmonic generation microscopy of partially oriented fiber structures

Second harmonic generation (SHG) in biological tissue originates predominantly from noncentrosymmetric fibrillar structures partially oriented within the focal volume (voxel) of a multiphoton excitation microscope. The study is aimed to elucidate fibrillar organization factors influencing SHG intensity, as well as achiral, R, and chiral, C, nonlinear susceptibility tensor component ratios. SHG response is calculated for various configurations of fibrils in a voxel using digital nonlinear microscope. The R and C ratios are calculated using linear incident and outgoing polarization states that simulate polarization-in polarization-out (PIPO) polarimetric measurements. The investigation shows strong SHG intensity dependence on parallel/antiparallel fiber organization. The R and C ratio is strongly influenced by the fiber chirality, tilting of the fibers out of image plane and crossing of the fibers. The study facilitates interpretation of polarimetric SHG microscopy images in terms of ultrastructural organization of fibers in the imaged structures. Statement of SignificanceSecond harmonic generation microscopy is widely used for imaging non-centrosymmetric biological structures such as collagen. The ultrastructure of collagen can be determined with polarimetric SHG microscopy. The coherent nonlinear response of biological structures depends on the 3D orientations and positions of the collagen fibers in the focal volume of the microscope. Here, we show how different fiber organizations and 3D orientations in the focal volume can affect the polarimetric SHG responses. The results are important for understanding and interpreting images obtained with polarimetric SHG microscopy.