Optimising Shifted Excitation Raman Difference Spectroscopy (SERDS) for application in highly fluorescent biological samples, using fibre optic probes†

Fibre optic probe based Raman spectroscopy can deliver in vivo molecular compositional analysis of a range of diseases. However, some biological tissues exhibit high levels of fluorescence which limit the utility of the technique, particularly when the fluorescence induces CCD etaloning, which can be particulalry hard to remove in subsequent analysis. Furthermore, use of fibre probes can result in silica signals superimposed on the biological Raman signals. Shifted excitation Raman difference spectroscopy (SERDS) utilises a small seperation in excitation wavelengths to remove signals from fluorescence, room lights, optical components and etaloning contributions, while retaining chemical signals from the sample. In this study, we sought to measure the optimum SERDS spectra enabling reconstruction of a range a narrow and broad peaks found in biological samples.  A original wavelength of 830 nm was utilised with 7 different shifts between 0.4 and 3.9 nm to determine which gave the best performance. This range roughly corresponds to the typical range of peak widths within biological Raman spectra at 830 nm excitation; 0.41 – 3.25 nm or 6 – 47 cm⁻¹. An wavelength shift of 2.4 nm was identified as optimal. Finally, a fibre optic Raman probe was used to measure 2 human lymph nodes ex vivo to demonstrate the feasibility of the approach with real-world examples.