Compound imaging of ocular tissues with backward second harmonic generation and two-photon excitation fluorescence

We report the implementation of compound imaging with backward second harnionic generation (SHG) and two-photon excitation fluorescence (TPEF) in a standard confocal microscope. Various biological tissues, including skin, muscle and ocular tissues (e.g. cornea. iris, choroid, and sclera) have been investigated with different sample preparation methods (fresh, fixation). The results show that collagen-rich ocular tissues, like cornea, iris, choroids, and sclera can produce strong SHG signals. In sclera, there are not only plenty of collagen, but also various endogenous fluorophores. The organization of collagen fibers in sclera is clearly distinguishable from its SHG images viewed transversely and horizontally respectively. We also find that fixation of the sample with formaldehyde has reduced the intensity of SHG intensity by almost 5O%. As the SHG and TPEF can provide complementary information about collagen and fluorophores rich biological tissues, compound imaging of SHG and TPEF presented in this study is believed to have potential applications in biomedicine and clinical diagnosis.