Polarization‐resolved SHG imaging as a fast screening method for collagen alterations during aging: Comparison with light and electron microscopy

Our previous study on rat skin showed that cumulative oxidative pressure induces profound structural and ultrastructural alterations in both rat skin epidermis and dermis during aging. Here, we aimed to investigate the biophotonic properties of collagen as a main dermal component in the function of chronological aging. We used second harmonic generation (SHG) and two‐photon excited fluorescence (TPEF) on 5 μm thick skin paraffin sections from 15‐day‐, 1‐month‐ and 21‐month‐old rats, respectively, to analyze collagen alterations, in comparison to conventional light and electron microscopy methods. Obtained results show that polarization‐resolved SHG (PSHG) images can detect collagen fiber alterations in line with chronological aging and that this method is consistent with light and electron microscopy. Moreover, the β coefficient calculated from PSHG images points out that delicate alterations lead to a more ordered structure of collagen molecules due to oxidative damage. The results of this study also open the possibility of successfully applying this fast and label‐free method to previously fixed samples. To counteract oxidative damage, aged collagen in rat dermis, becomes more ordered and aligned. β coefficient calculation from polarization‐resolved SHG images is excellent fast screening tool for studying this fine alterations at the molecular level. Comparative study was performed on dermis of 15‐days‐, 1‐month‐ and 21‐months‐old rats using conventional light and advanced TPEF and SGH microscopy. Transmission electron microscopy revealed ultrastructural backgrounds of higher‐ordered collagen fibers due to peroxidation and nitration.