Online Fluorescence Suppression in Modulated Raman Spectroscopy

Abstract

Label-free chemical characterization of single cells is an important aim for biomedical research. Standard Raman spectroscopy provides intrinsic biochemical markers for noninvasive analysis of biological samples but is often hindered by the presence of fluorescence background. In this paper, we present an innovative modulated Raman spectroscopy technique to filter out the Raman spectra from the fluorescence background. The method is based on the principle that the fluorescence background does not change whereas the Raman scattering is shifted by the periodical modulation of the laser wavelength. Exploiting this physical property and importantly the multichannel lock-in detection of the Raman signal, the modulation technique fulfills the requirements of an effective fluorescence subtraction method. Indeed, once the synchronization and calibration procedure is performed, minimal user intervention is required, making the method online and less time-consuming than the other fluorescent suppression methods. We analyze the modulated Raman signal and shifted excitation Raman difference spectroscopy (SERDS) signal of 2 mum-sized polystyrene beads suspended in a solution of fluorescent dye as a function of modulation rate. We show that the signal-to-noise ratio of the modulated Raman spectra at the highest modulation rate is 3 times higher than the SERDS one. To finally evaluate the real benefits of the modulated Raman spectroscopy, we apply our technique to Chinese hamster ovary cells (CHO). Specifically, by analyzing separate spectra from the membrane, cytoplasm, and nucleus of CHO cells, we demonstrate the ability of this method to obtain localized sensitive chemical information from cells, away from the interfering fluorescence background. In particular, statistical analysis of the Raman data and classification using PCA (principal component analysis) indicate that our method allows us to distinguish between different cell locations with higher sensitivity and specificity, avoiding potential misinterpretation of the data obtained using standard background procedures.

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.…”

Spectral pre-processing for biomedical vibrational spectroscopy and microspectroscopic imaging

“…Although Raman spectroscopy has been proven to be a powerful tool for biomedical and microbiological applications it has been severely limited in its applicability by fluorescence [51]. Fluorescence is characterized as a broad band emission that occurs in the same wavelength interval as the Raman signal.…”

“…The fluorescence signal can be also separated by using anti-Stokes Raman spectroscopy [59] or by employing different polarization properties of fluorescence and Raman scattered light [60]. The next option for fluorescence rejection is known as shifted excitation Raman difference spectroscopy (SERDS) which makes use of the fact, that the fluorescence background does not change whereas the Raman scattering is frequency-shifted when the laser excitation wavelength is periodically modulated [51,61,62]. The third group of methods comprises purely mathematical or chemometric techniques to mitigate the fluorescence components of Raman spectra.…”

Enhanced bioanalyte detection in waveguide confined Raman spectroscopy using wavelength modulation

“…Numerous methods have been proposed to reduce or suppress the fluorescence background [13][14][15][16][17][18]. Here, we use a continuous wavelength modulation technique that consists in periodically changing the excitation wavelength thereby distinguishing between the static background and the modulated Raman peaks [19]. Additionally, the uses of the continuous wavelength modulation enhances the Raman Signal to Noise Ratio (SNR) compared to the standard shifted excitation Raman spectroscopic methods [20].…”