## 3 Results and Discussion
A comparison of the loading plots of the PCs most involved in discrimination in Figure 2a,b with the difference in mean spectra could provide useful information regarding the Raman signals responsible for the spectral differences between the two classes of spectra. The good matching of loading plots and difference spectra is clearly visible in Figure 2c,d. In particular, the loading plot of nucleus spectra presents positive peaks at about 1250, 1340 and 1580 cm\({}^{-1}\) and negative peaks at about 1060, 1450 and 1650 cm\({}^{-1}\). In contrast, the loading plot of cytoplasm spectra has positive peaks at about 1060, 1440 and 1650 cm\({}^{-1}\) and negative peaks at about 1250 and 1335 cm\({}^{-1}\). The attribution of Raman signals at the above spectral positions (as shown in Table 1) confirms that the differences between healthy and cancer cells is mainly due to the relative contents of lipids and nucleic acids. Specifically, lipid content is higher in healthy cells (as specified by the negative peak at 1060 cm\({}^{-1}\) in Figure 2c and positive peak at 1450 cm\({}^{-1}\) in Figure 2d), while nucleic acid content is higher in cancer cells (as suggested by the positive peaks at 1340 and 1580 cm\({}^{-1}\) in Figure 2c). Similar findings were also reported by us [14] and other authors by means of Raman [22; 23] and FTIR [24] measurements of colon cells and tissue. Instead, contradictory results are obtained about the relative protein content; indeed, each loading plot shows protein-related signals of opposite sign (at 1250, 1450 and 1650 cm\({}^{-1}\) in Figure 2c and at 1250, 1335, 1650 cm\({}^{-1}\) in Figure 2d). Such a result is in disagreement with that reported by \begin{table}
\begin{tabular}{c c} \hline
**Spectral Position (cm\({}^{-1}\))** & **Assignment** \\ \hline
1004 & C-C symmetric ring breathing of phenylalanine (p.) \\
1031 & C-H in plane bending of phenylalanine (p.) \\
1064 & C-C stretching (L) \\
1088 (cytoplasm spectra) & C-N stretching (p.) and C-C stretching (l.) \\
1097 (nucleus spectra) & Symmetric PO\({}_{2}\) stretching of DNA (n.a.) \\
1129 & C-N stretching (p.)-C stretching (c.) and C-C stretching (l.) \\
1174 & C-H bending amino acids (p.) \\
1210 & C-C\({}_{6}\)H stretching amino acids (p.) \\
1250 & Amide III (p.) \\
1272 & \multirow{2}{*}{CH\({}_{3}\)CH\({}_{2}\) wagging mode in purine bases of DNA (n.a.) and collagen (p.)} \\
1326 & & \\
1340 & & \\
1406 & & \\
1450 & & \\
1580 & Ring breathing modes in DNA bases (n.a.) \\
1657 & Amide I (p.) \\ \hline \end{tabular}
\end{table} 
Table 1: Attribution of Raman peaks according to the literature results [19]. Abbreviations: p.—proteins; l.—lipids; n.a.—nucleic acids. other authors, who measured more intense protein Raman peaks in normal cells compared to cancerous ones [22; 25]. The explanation for this disagreement could be due to the fact that each of the two cell lines is characterized by its own Raman spectrum with its own characteristic relative peak intensities. In particular, the relative intensities of protein peaks in the spectra of CaCo-2 cells are larger than the relative intensities of the corresponding peaks in FHC cells. They do not necessarily have to be different, because CaCo-2 cells do not correspond to FHC cells that have evolved into a cancerous state (and therefore the intensities of some protein Raman peaks should decrease). Despite this disagreement with the literature reports, it is important to remark that a change in the relative intensity of some Raman peaks (as occurs in the evolution of a cell from a healthy to a cancerous state) can be detected by Raman spectroscopy and PCA analysis. Therefore, PCA promotes the possibility of classification of spectra of unknown nature by means of proper multivariate techniques, based on the simultaneous use of whole Raman spectra (instead of single Raman peaks). The obtained results suggest that a PCA-based classification model should be developed that allows unknown spectra to be assigned to the right class.