Polarization‐dependent second‐harmonic generation (P‐SHG) microscopy is used to characterize molecular nonlinear optical properties of collagen and determine a three‐dimensional (3D) orientation map of collagen fibers within a pig tendon. C6 symmetry is used to determine the nonlinear susceptibility tensor components ratios in the molecular frame of reference χzzz2/χzxx2 and χxyz2/χzxx2, where the latter is a newly extracted parameter from the P‐SHG images and is related to the chiral structure of collagen. The χxyz2/χzxx2 is observed for collagen fibers tilted out of the image plane, and can have positive or negative values, revealing the relative polarity of collagen fibers within the tissue. The P‐SHG imaging was performed using a linear polarization‐in polarization‐out (PIPO) method on thin sections of pig tendon cut at different angles. The nonlinear chiral properties of collagen can be used to construct the 3D organization of collagen in the tissue and determine the orientation‐independent molecular susceptibility ratios of collagen fibers in the molecular frame of reference.

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