A shifted-excitation Raman difference spectroscopy (SERDS) evaluation strategy for the efficient isolation of Raman spectra from extreme fluorescence interference

A biochemical characterization of pathologies in biological tissue can be provided by Raman spectroscopy. Often, the raw spectrumis severely affected by fluorescence interference. We report and compare various spectra-processing approaches requiredfor the purification of Raman spectra from heavily fluorescence-interfered raw spectra according to the shifted-excitation Ramandifference spectroscopy method. These approaches cover the entire spectra-processing chain fromthe raw spectra to the purifiedRaman spectra. In detail, we compared (1) area normalization versus z-score normalization, (2) direct reconstruction of the differencespectra versus reconstruction of zero-centered difference spectra and (3) collective baseline correction of the reconstructedspectra versus piecewise baseline correction of the reconstructed spectra and, finally, (4) analyzed the influence of the shift of theexcitation wavelength on the quality of the reconstructed spectra. Statistical analysis of the spectra showed that – in our experiments– the best results were obtained for the z-score normalization before subtraction of the normalized spectra, followed byzero-centering of the difference spectra before reconstruction and a piecewise baseline correction of the pure Raman spectra.Withour equipment, a wavelength shift from 784 to 785 nm provided reconstructed spectra of best quality. The analyzed specimenswere different tissue types of pigs, tissue from the oral cavity of humans and a model solution of dye dissolved in ethanol.