Effective Rejection of Fluorescence Interference in Raman Spectroscopy Using a Shifted Excitation Difference Technique

Resonance Raman spectroscopy is a powerful technique for probing the vibrations of particular chromophores in multicomponent systems. By tuning the wavelength of the excitation laser into resonance with an electronic absorption band of only one molecular species, the vibrational Raman scattering from this species can be selectively enhanced. Thus, resonance Raman spectroscopy can provide structural information for chromophores in solution or biological chromophores within their functionally active protein environment. However, since the very nature of the experiment requires that the excitation light be absorbed by the sample, the measurement of resonance Raman spectra is often made difficult by a large fluorescence background in the same spectral region as the Raman scattering. The problem of fluorescence interference from intrinsic sample emission is often further exacerbated in biological samples, where low-concentration impurities with large fluorescence yields can be difficult to remove. Even weak fluorescence, with an effective fluorescence quantum yield of ≈10−4, completely overwhelms resonance Raman signals, which have typical quantum yields of ≈10−7.