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

• Published in: Journal of biophotonics 2015-09, Vol.8 (9), p.705-713
• Main Authors: Segawa, Hiroki, Kaji, Yuichi, Leproux, Philippe, Couderc, Vincent, Ozawa, Takeaki, Oshika, Tetsuro, Kano, Hideaki
• Format: Article
• Language: English
• Subjects: Animals; Bioengineering; Coherent scattering; Cornea; Cornea - chemistry; Cornea - cytology; Engineering Sciences; Imaging; Lasers; Life Sciences; Light; Male; molecular imaging; Molecular structure; multimodal imaging; Multimodal Imaging - instrumentation; Multiplexing; Nonlinear Sciences; Nonlinearity; Optics; Photonic; Photons; Raman scattering; Rats; Spectra; Spectrum Analysis; spectrum analysis Raman/instrumentation; Vibration
• ISSN: 1864-063X; 1864-0648
• DOI: 10.1002/jbio.201400059

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. Multimodal nonlinear spectral imaging with coherent anti‐Stokes Raman scattering (CARS) molecular fingerprinting was applied to ex vivo measurement of rat cornea. Complicated inner structure of the cornea was clearly visualized with molecular structural information produced by combined spectral analysis of multiplex CARS, second harmonic generation (SHG) and multiplex third‐order sum frequency generation (TSFG). This study will open the door for molecular level analysis in corneal medical diagnostics.