Polarimetric second-harmonic generation microscopy of partially oriented fibers I: Digital modeling

Second-harmonic generation (SHG) in biological tissues originates predominantly from noncentrosymmetric fibrillar structures partially oriented within a focal volume (voxel) of a multiphoton excitation microscope. This 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 the digital nonlinear microscope. The R and C ratios are calculated using linear incident and outgoing polarization states that simulate polariza-tion-in polarization-out polarimetric measurements. The investigation shows strong SHG intensity dependence on parallel/anti-parallel fiber organization. The R and C ratios are strongly influenced by the fiber chirality, tilting of the fibers out of the image plane, and crossing of the fibers. The computational modeling provides the basis for the interpretation of polarimetric SHG mi-croscopy images in terms of the ultrastructural organization of fibers in each voxel of the samples. The modeling results are em-ployed in the accompanying paper to investigate the ultrastructures with parallel/antiparallel fibers and two-dimensional and tree-dimensional crossing fibers in biological and biomimetic structures. SIGNIFICANCE Second-harmonic generation microscopy is widely used for imaging noncentrosymmetric 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 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 response. The results are important for understanding and interpreting images obtained with polarimetric SHG microscopy.

Automated summary

This study aims to elucidate the factors affecting the intensity of second-harmonic generation (SHG) in biological tissues and the ratios of achiral (R) and chiral (C) nonlinear susceptibility tensor components. The results show a strong dependence of SHG intensity on the organization of parallel/anti-parallel fibers, as well as the fiber chirality, tilting, and crossing. The computational modeling provides a basis for interpreting polarimetric SHG microscopy images.