We have demonstrated molecular spectral imaging based on high speed SRS (Raman scattering microscopy) spectral microscopy and modified ICA (independent component analysis). This system allows quick image acquisition at.30 frames/s by changing the wavenumber in a frame-by-frame manner. The modified ICA enables blind separation of constituents without a priori spectral data. The IC spectra give spectroscopic information, and the IC images can be used to produce multi colour images. This label-free and rapid observation method would be especially useful in medical imaging. We present various imaging modalities such as two-dimensional spectral imaging of rat liver, two-colour three-dimensional imaging of a vessel in rat liver, spectral imaging of several sections of intestinal villi in mouse, and in vivo spectral imaging of mouse ear skin.

In SRS microscopy, we use two-color laser pulses, one of which is intensity modulated in time. Then they are focused on a sample. When the optical frequency difference of the pulses matches the vibrational frequency of the sample molecules, the optical energy of the high-frequency pulse is transferred to the low-frequency pulse and to molecular vibrations through the SRS process. As a result, the intensity modulation is transferred to the other pulse, and this modulation transfer is measured by the lock-in detection technique. SRS has various advantages, such as accessibility to the vibrational spectrum without spectral distortion, quantitative image contrast, high sensitivity, and high imaging speed.