Polarization-resolved hyperspectral stimulated Raman scattering microscopy for label-free biomolecular imaging of the tooth

We report the development and implementation of a rapid polarization-resolved hyperspectral stimulated Raman scattering (SRS) microscopy technique for label-free biomolecular imaging of the tooth. The hyperspectral SRS imaging technique developed covers both fingerprint (800–1800 cm−1) and high-wavenumber (2800–3600 cm−1) regions for tooth Raman imaging without fluorescence background interference with an imaging speed of <0.3 s per frame of 512 × 512 pixels (∼1 μs per pixel), that is, >106 faster than confocal Raman imaging. Significant differences of hyperspectral SRS spectra among different tooth locations (e.g., dentin, enamel, and dentin-enamel junction) are observed, revealing the biochemical distribution differences across the tooth. Further polarization-resolved SRS imaging shows different polarization dependences related to the molecular orientation differences of various tooth locations. This work demonstrates the potential of polarization-resolved hyperspectral SRS imaging technique developed in rapidly characterizing biochemical structures and compositions as well as biomolecule organizations/orientations of the tooth without labeling.