Multi-excitation Raman spectroscopy technique for fluorescence rejection

Multi-excitation Raman spectroscopy filters out Raman signals from a fluorescent background by sequentially using multiple excitation frequencies. The filtering method exploits the shift of the Raman spectra with excitation frequency and the static response of the fluorescent background. This technique builds upon previous work which used two slightly shifted excitations, Shifted Excitation Raman Difference Spectroscopy (SERDS), in order to filter the Raman signal. An Expectation-Maximization algorithm is used to estimate the Raman and fluorescence signals from multiple spectra acquired with slightly shifted excitation frequencies. In both simulation and experiment, the efficacy of the algorithm increases with the number of excitation frequencies even when holding the total excitation energy constant, such that the signal to noise ratio is inversely proportional to the number of excitation frequencies. In situations where the intense fluorescence causes significant shot noise compared to the weak Raman signals, the multi-excitation approach is more effective than non-iterative techniques such as polynomial background subtraction.