Simple Reconstruction Algorithm for Shifted Excitation Raman Difference Spectroscopy

Raman spectroscopy is often hampered by high fluorescence backgrounds that can easily swamp the much weaker Raman signals. A common solution to avoid this is to use excitation wavelengths that do not excite fluorescence, for example, using near-infrared (NIR) sources that operate in a region where few molecular systems have electronic absorption bands. However, while this circumvents the fluorescence problem very effectively,1–3 it fails to take advantage of the unique features offered by the resonance Raman technique in terms of tremendous sensitivity and selectivity that are so beneficial for investigating complex heterogeneous samples such as those encountered in many biomedical applications. One of the most widely used approaches for obtaining Raman data from molecular systems that give large fluorescence backgrounds is shifted excitation Raman difference spectroscopy4–6 (SERDS), a technique that is popular for its technical simplicity. The key benefit of SERDS is that it can effectively eliminate the large fluorescence backgrounds as well as other sources of random or systematic noise generated by, for example, the detector where the different sensitivity of individual pixels of the detector can produce systematic effects.5 Application of SERDS has permitted great sensitivity down to true photon shot levels.4 In its basic form, the SERDS approach relies on obtaining two Raman spectra using excitation wavelengths shifted by an amount comparable to the bandwidth of the measured Raman bands (typically 5–10 cm21). A similar but less effective result can also be achieved by shifting the spectrograph spectral window by this amount, and this can be preferred because of its instrumental simplicity.5,6 Having obtained two Raman spectra, S9l and S9l2d, these are subtracted from each other to produce a difference spectrum, D 5 S9l 2 S9l2d. This step results in the elimination of the invariant fluorescence signal that for the small change in excitation wavelength is unchanged. The invariance is a direct result of the fact that the majority of fluorescence is emitted from vibrationally relaxed states (Kasha’s rule) and a small shift in the excitation wavelength does not perturb its spectral profile. In