Multivariate methodology for surface enhanced Raman chemical imaging of lymphocytes

Surface enhanced Raman spectroscopy (SERS) was used to study the uptake of rhodamine 6G in human lymphocytes. In total four Raman images of lymphocytes were used. The aim was to find a multivariate methodology capable of separating spectra with chemical information from those that mainly contained the surface enhanced background, in order to create chemical images. The standard PCA procedure was compared with PCA of standard normal variate (SNV) corrected spectra, spectra baseline corrected in the wavelet domain, and variable trimming before PCA, to isolate unique spectra. It was not straightforward to perform a standard PCA for overview, since the small background variation in many variables dominated over the Raman band variation that only occur in few variables. It was shown that wavelet filtering could remove background variations and that variable trimming followed by PCA modelling left the unique Raman spectra as outliers, which facilitated interpretation of the Raman score images.