## 3 Results and Discussion  
The comparison between the SNV-normalized spectra of FHC and CaCo-2 cells from the calibration set is shown in Figure 1. In particular, the mean (continuous lines) and standard deviation (dashed lines) spectra are displayed for both LWR and HWR. Since the two mean spectra are almost overlapping, they have been intensity-shifted in Figure 1 for clarity. These spectra are similar to those reported for colon cells and tissues by other authors [8; 15]. They are characterized by several spectral peaks, which can be related to the IR radiation absorption from specific functional groups inside the main biochemical cellular components. Specifically, the most evident and resolved peaks (labelled in Figure 1) in the LWR were due to absorption from nucleic acids and from protein and lipid groups, whereas the HWR was dominated by absorption from protein and lipid components [42]. The standard deviation values in Figure 1 emphasize that the absorption signals of healthy cells are more broadly distributed with respect to those of cancerous cells, suggesting that healthy cells present larger differences in the relative content of cellular components with respect to the cancerous ones. In addition, we remark that no baseline was subtracted to the spectra during the pre-processing step, because the analytical function corresponding to the scattering signal, which is mainly responsible for the background, was unknown. Thus, the hypotheses we could make regarding this could be unreliable, and, consequently, they could influence the spectra in an arbitrary way. The increasing and decreasing trends of the spectral intensity signals in the LWR and HWR, respectively, suggest that a baseline signal was still present. Therefore, the SNV normalization failed to totally remove the scattering signal. However, the similar trends of the standard deviation curves indicate that the scattering contribution is comparable for both spectral ranges of the two cell types; therefore, we believe that the incomplete removal of the scattering signals does not drastically influence the spectral analysis. In order to correctly identify the spectral position of the absorption peaks which mainly contribute to the FTIR spectra, the second derivative signal of the mean spectrum was calculated and is reported in Figure 2 (red line), as far as the healthy cells are concerned. In fact, second-order derivatives are characterized by negative bands with minima at the spectral position corresponding to maxima on the zero-order bands (as indicated by the dot-dashed lines). Therefore, the spectral positions of minima in the second derivative spectrum can be assumed to correspond to the spectral positions of single FTIR absorption peaks. Each of such absorption peaks is related to the contribution of specific functional groups inside the cellular components: the assignment of the absorption peaks is reported in Table 1, as was deduced in [42]. Figure 1: Mean FTIR spectra of healthy FHC (continuous black line) and cancerous CaCo-2 (continuous blue line) cells of the calibration set after SNV normalisation. Standard deviation spectra are also reported as dashed lines. The assignment of some evident vibrational peaks to cell components is also reported. The spectra have been vertically shifted for clarity. The absorption values of several selected features are partially able to differentiate healthy from cancerous cells, as shown in Figure 3. In particular, the absorption intensity values at 1740 cm\({}^{-1}\) and 2921 cm\({}^{-1}\) were larger in healthy cells than in cancerous ones, as evident in Figure 3a,b. This observation suggests that the healthy cells have a larger relative amount of lipids with respect to the cancerous cells. The greater intensity of lipid absorption peaks in the normal samples than in the cancerous ones was also reported by L.