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Raman spectroscopy has demonstrated great potential in biomedical applications. However, spectroscopic Raman imaging is not widely used because of slow data acquisition. Our previous studies have indicated that spectroscopic Raman imaging can be significantly sped up using the approach of narrow-band imaging followed by spectral reconstruction. A multi-channel system has been built to demonstrate the feasibility of fast wide-field Raman spectroscopic imaging based on simultaneous narrow-band image acquisition and spectral reconstruction based on Wiener estimation in phantoms. To further improve the accuracy of reconstructed Raman spectra, we propose a stepwise spectral reconstruction method in this study, which can be combined with the earlier developed sequential weighted Wiener estimation to improve the spectral reconstruction accuracy. The stepwise spectral reconstruction method first reconstructs the fluorescence background spectra from narrow-band measurements by sequential weighted Wiener estimation and then the pure Raman narrow-band measurements can be estimated by subtracting the estimated fluorescence background from the overall Raman measurements. Thereafter, pure Raman spectra can be reconstructed from estimated pure Raman narrow-band measurements. The result indicates that the stepwise spectral reconstruction method can improve the spectral reconstruction accuracy by more than 30% when combined with sequential weighted Wiener estimation, compared with traditional Wiener estimation. In addition, cell Raman imaging were realized by using a multi-channel wide field Raman spectroscopic imaging and the stepwise spectral reconstruction method. This method can potentially facilitate the use of spectroscopic Raman imaging to investigate fast changing phenomena in biological samples.
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