A method is proposed for automatically determining the upper and lower boundaries of the Raman scatter peak in fluorescence spectroscopy from empirical data. Accurate peak boundaries are needed to calculate accurate Raman peak areas, used for normalizing fluorescence signals to produce data in units that are comparable between instruments. Comparisons of Raman peak boundaries across nine individual instruments (FluoroMax 3 (FM3) fluorometers from HORIBA Jobin Yvon and Cary Eclipse (CE) fluorometers from Varian Inc.) at the excitation wavelength λ<sub>ex</sub> = 350 nm reveal consistent results. At 350 nm excitation, the Raman peak was confined by the emission wavelengths of 382–418 nm, with boundaries determined for the FM3 fluorometers deviating by no more than 0.5 nm and 1.5 nm with respect to the start and end of the peak, and CE fluorometers deviating by up to 1.5 nm and 2 nm, respectively. Peak width was a function of fluorometer type and excitation wavelength. For the FM3 instruments, widths increased from approximately 30 nm at λ<sub>ex</sub> = 300 nm to 40 nm at λ<sub>ex</sub> = 380 nm, while for the CE instruments, peaks were approximately 5–8 nm narrower. Code for implementing the procedure in MATLAB, which allows for the adjustment of input parameters to compensate for noisy data, is provided in the Supplemental Material (available online).
Vol. 6, Iss. 3 Virtual Journal for Biomedical Optics
Kathleen R. Murphy, "A Note on Determining the Extent of the Water Raman Peak in Fluorescence Spectroscopy," Appl. Spectrosc. 65, 233-236 (2011)