Automated Method for Subtraction of Fluorescence from Biological Raman Spectra

Abstract

One of the challenges of using Raman spectroscopy for biological applications is the inherent fluorescence generated by many biological molecules that underlies the measured spectra. This fluorescence can sometimes be several orders of magnitude more intense than the weak Raman scatter, and its presence must be minimized in order to resolve and analyze the Raman spectrum. Several techniques involving hardware and software have been devised for this purpose; these include the use of wavelength shifting, time gating, frequency-domain filtering, first- and second-order derivatives, and simple curve fitting of the broadband variation with a high-order polynomial. Of these, polynomial fitting has been found to be a simple but effective method. However, this technique typically requires user intervention and thus is time consuming and prone to variability. An automated method for fluorescence subtraction, based on a modification to least-squares polynomial curve fitting, is described. Results indicate that the presented automated method is proficient in fluorescence subtraction, repeatability, and in retention of Raman spectral lineshapes.

Load-bearing in cortical bone microstructure: Selective stiffening and heterogeneous strain distribution at the lamellar level

“…The Fluorescent background was removed from all raw spectra in MATLAB using a modified 5 th order polynomial fit algorithm (Lieber and Mahadevan-Jansen, 2003); spectra were then smoothed for noise reduction and the intensity or the ratio of userdefined Raman peaks were measured. Finally, ȝ-Raman compositional images were reconstructed by assigning a grey level value to the measured intensity or ratio value (Figure 1 -a).…”

Bone material properties in actively bone-forming trabeculae in postmenopausal women with osteoporosis after three years of treatment with once-yearly Zoledronic acid

“…Once acquired, the Raman spectra were baseline corrected (rubber band, five iterations) so as to account for fluorescence, (10) and the following Raman parameters were calculated, as published elsewhere (2,11) : (1) The mineral/matrix ratio was expressed as the integrated areas of the v 1 PO 4 (930 to 980 cm À1 ) to the amide I (1620 to 1700 cm À1 ) bands and of Crosses indicate regions where Raman spectra were acquired. For this study, only spectra obtained between the two labels (denoted by black arrows) were processed.…”

Spectral pre-processing for biomedical vibrational spectroscopy and microspectroscopic imaging

“…The third group of methods comprises purely mathematical or chemometric techniques to mitigate the fluorescence components of Raman spectra. Such methods include (fluorescence) baseline subtraction procedures using polynomial fittings [63][64][65], the use of first or second derivative filters [52,66], the shifted-spectra technique [52], PCA analysis [67], wavelet transformations [68,69], and the application of FT frequency filters [25, 52,]. Though each of these methods has been shown to be useful in certain situations, they are not without limitations.…”