Optimal algorithm for fluorescence suppression of modulated Raman spectroscopy

Abstract

Raman spectroscopy permits probing of the molecular and chemical properties of the analyzed sample. However, its applicability has been seriously limited to specific applications by the presence of a strong fluorescence background. In our recent paper [Anal. Chem. 82, 738 (2010)], we reported a new modulation method for separating Raman scattering from fluorescence. By continuously changing the excitation wavelength, we demonstrated that it is possible to continuously shift the Raman peaks while the fluorescence background remains essentially constant. In this way, our method allows separation of the modulated Raman peaks from the static fluorescence background with important advantages when compared to previous work using only two [Appl. Spectrosc. 46, 707 (1992)] or a few shifted excitation wavelengths [Opt. Express 16, 10975 (2008)]. The purpose of the present work is to demonstrate a significant improvement of the efficacy of the modulated method by using different processing algorithms. The merits of each algorithm (Standard Deviation analysis, Fourier Filtering, Least-Squares fitting and Principal Component Analysis) are discussed and the dependence of the modulated Raman signal on several parameters, such as the amplitude and the modulation rate of the Raman excitation wavelength, is analyzed. The results of both simulation and experimental data demonstrate that Principal Component Analysis is the best processing algorithm. It improves the signal-to-noise ratio in the treated Raman spectra, reducing required acquisition times. Additionally, this approach does not require any synchronization procedure, reduces user intervention and renders it suitable for real-time applications.

Raman spectroscopy: techniques and applications in the life sciences

“…Dholakia and his group developed a technique called Modulated Raman Spectroscopy (MRS) [170,171]. MRS is achieved by modulating the excitation wavelength.…”

Modulated Raman spectroscopy for enhanced identification of bladder tumor cells in urine samples

“…Indeed, higher modulation frequencies decrease the 1/f noise level while increasing the shot noise. 16 To evaluate the SR and MR spectra of control and urinestressed HUC and MGH cells and to compare different cell classes against each other, PCA 21 was carried out. The specificity/sensitivity and, more generally, the confusion matrix was determined using a leave-one-out procedure in which each spectrum is classified against the PCA of all the other spectra.…”

“…15,16 The method is based on the wavelength shift of the Raman peaks as the wavelength of the excitation laser is continuously modulated while the fluorescence background remains static. By using an algorithm based on principal component analysis (PCA) of the raw data, mostly fluorescence-free Raman spectra are obtained.…”

Label-free haemogram using wavelength modulated Raman spectroscopy for identifying immune-cell subset

“…The entire single cell Raman measurements were performed on a confocal Raman system based arranged with an inverted microscope to obtain Raman signatures from the immune cell subsets. WMRS was used to suppress the contribution of the fluorescence background in the Raman signature, resulting in differential Raman spectra being obtained [15]. The Raman signal was recorded from each of the isolated immune cell subsets with an acquisition time of 25 s for an individual cell.…”