High-Speed 3D Spectral Imaging with Stimulated Raman Scattering

Stimulated Raman scattering (SRS) microscopy is a powerful technique for label-free biomedical imaging in real time and in 3D. Indeed, SRS provides chemical contrast of sample molecules reflecting molecular vibrations in a quantitative manner. However, in previous SRS microscopy, it has been difficult to discriminate different molecules with overlapping Raman bands since SRS microscopy visualizes molecular vibrations at a single frequency, which is determined by the optical frequency difference between two-color excitation laser pulses. In order to improve the molecular specificity of SRS microscopy, it will be advantageous to conduct spectral imaging, where SRS images at various Raman shifts are acquired. This chapter reviews the current status of SRS microscopy and discusses how spectral imaging can be accomplished in SRS microscopy. Then the recent development of our high-speed SRS spectral microscopy is introduced. This system enables video-rate SRS imaging while the vibrational frequency is varied in a frame-by-frame manner. Furthermore, in order to discriminate different constituents, spectral images are processed by modified independent component analysis. Spectral imaging of biological tissues in 3D is demonstrated.