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

• Published in: Journal of Raman spectroscopy Vol. 47; no. 2; pp. 198 - 209
• Main Authors: Gebrekidan, Medhanie Tesfay, Knipfer, Christian, Stelzle, Florian, Popp, Juergen, Will, Stefan, Braeuer, Andreas
• Format: Journal Article
• Language: English
• Published: Bognor Regis Blackwell Publishing Ltd 01-02-2016; Wiley Subscription Services, Inc
• Subjects: Biological; Fluorescence; fluorescence rejection; Raman spectra; Raman spectroscopy; Raw; Reconstruction; reconstruction of Raman spectra; shifted-excitation Raman difference spectroscopy; Spectra; Spectroscopy
• ISSN: 0377-0486; 1097-4555
• DOI: 10.1002/jrs.4775

A biochemical characterization of pathologies in biological tissue can be provided by Raman spectroscopy. Often, the raw spectrum is severely affected by fluorescence interference. We report and compare various spectra‐processing approaches required for the purification of Raman spectra from heavily fluorescence‐interfered raw spectra according to the shifted‐excitation Raman difference spectroscopy method. These approaches cover the entire spectra‐processing chain from the raw spectra to the purified Raman spectra. In detail, we compared (1) area normalization versus z‐score normalization, (2) direct reconstruction of the difference spectra versus reconstruction of zero‐centered difference spectra and (3) collective baseline correction of the reconstructed spectra versus piecewise baseline correction of the reconstructed spectra and, finally, (4) analyzed the influence of the shift of the excitation 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 by zero‐centering of the difference spectra before reconstruction and a piecewise baseline correction of the pure Raman spectra. With our equipment, a wavelength shift from 784 to 785 nm provided reconstructed spectra of best quality. The analyzed specimens were different tissue types of pigs, tissue from the oral cavity of humans and a model solution of dye dissolved in ethanol. © 2015 The Authors. Journal of Raman Spectroscopy published by John Wiley & Sons Ltd.
In order to apply Raman spectroscopy for biochemical characterization of pathologies in biological tissue, we report and compare various spectra‐processing approaches required for the purification of Raman spectra from heavily fluorescence‐interfered raw spectra according to the shifted‐excitation Raman difference spectroscopy method. Firstly, the spectra are normalized and subtracted. Secondly, the difference spectrum is processed to minimize further the still‐left fluorescence. Then, reconstruction of the difference spectrum followed by piecewise baseline correction gives a purified Raman spectrum.