Multimodal and multiplex spectral imaging of rat cornea ex vivo using a white-light laser source.

We applied our multimodal nonlinear spectral imaging microscope to the measurement of rat cornea. We successfully obtained multiple nonlinear signals of coherent anti-Stokes Raman scattering (CARS), third-order sum frequency generation (TSFG), and second harmonic generation (SHG). Depending on the nonlinear optical processes, the cornea tissue was visualized with different image contrast mechanism simultaneously. Due to white-light laser excitation, multiplex CARS and TSFG spectra were obtained. Combined multimodal and spectral analysis clearly elucidated the layered structure of rat cornea with molecular structural information. This study indicates that our multimodal nonlinear spectral microscope is a promising bioimaging method for tissue study. Multimodal nonlinear spectral images of rat cornea at corneal epithelium and corneal stroma in the in-plane (XY) direction. With use of the combinational analysis of different nonlinear optical processes, detailed molecular structural information is available without staining or labelling.