## 3 Results and Discussion  
The mean and standard deviation spectra measured on the nucleus (a) and cytoplasm (b) region of healthy and cancer cells from the calibration set are reported in Figure 1. The spectral positions of the main Raman signals (labelled in Figure 1) correspond to vibrational modes detailed in Table 1, in agreement with the literature paper by [PERSON] et al. [19]. The spectral positions labelled in Figure 1 were obtained by calculating the second derivative signal of the mean spectra and considering the negative minima of such spectra. It is clearly evident that the spectra of both cell types are quite similar, as well as being similar to previously reported Raman spectra of colon cells [20,21] and tissues [22]. In particular, the spectra are dominated by the amide I peak at about 1660 cm\({}^{-1}\) and the CH\({}_{2}\) deformation peak at about 1450 cm\({}^{-1}\). Both these main peaks are related to vibrational modes in protein components. The protein components also contribute to the Raman spectra with the amide III peaks at 1250 and 1272 cm\({}^{-1}\), the phenylalanine ring breathing peak at 1004 cm\({}^{-1}\), the C-N stretching peaks at 1088 and 1129 cm\({}^{-1}\), and aromatic ring vibrations of several amino acids at 1031, 1174 and 1210 cm\({}^{-1}\). The Raman peaks related to nucleic acid components (mainly visible in the nucleus core spectra) are located at 1097 cm\({}^{-1}\) (PO\({}_{2}\) phosphodioxy bond), 1326 and 1340 cm\({}^{-1}\) (CH\({}_{3}\)/CH\({}_{2}\) wagging mode in the bases of DNA and ring mode of DNA/RNA bases, respectively), and 1580 cm\({}^{-1}\) (vibrational ring breathing mode of adenine and guanine). One well-resolved Raman peak corresponding to the lipid vibrational mode is found at 1064 cm\({}^{-1}\), due to the C-C stretching mode. However, other lipid peaks overlap with protein peaks at 1088, 1129 and 1450 cm\({}^{-1}\), mainly in the cytoplasm spectra. Figure 1: Mean Raman spectra of normalized healthy and cancerous colon cells. The spectra were measured within a cell volume including the nucleus (**a**) and excluding it (**b**). Standard deviation spectra are shown as dashed lines. The spectra have been vertically shifted. The labels indicate the spectral position of the main Raman peaks. The Raman signal values of most peaks are not very different for the spectra of the two cell types: consequently, the intensity value of a specific Raman peak does not reliably allow the identification of which class it belongs to. Therefore, multivariate analysis techniques (in which many spectral intensity values are considered) may be useful to study the possibility of discriminating healthy cells from cancer cells according to the measured Raman spectra. A proper multivariate technique able to estimate the possibility to discriminate spectra from two different groups on the basis of their Raman intensity values is PCA. In fact, although PCA is not a classification technique, it is a reliable method to identify differences between spectral data. Therefore, we analysed data from the calibration set by PCA, using a full cross-validation method. Figure 2a,b show the PCA score plot of PC2 vs. PC1 and PC3 vs. PC2, respectively, for the Raman spectra of the calibration set. The PC2 and PC3 score values discriminate the spectra of cancerous cells from those of healthy cells well, regardless of the cell region in which the spectra were measured. Indeed, Figure 2a points out that the healthy cells' spectra have mainly negative PC2 score values, whereas the cancerous cells have positive values. The opposite occurs for the PC3 score values of the cytoplasm spectra (Figure 2b). In particular, 10 and 6 principal components are needed to include 68% of all the variance of the spectra in the nucleus and cytoplasm calibration sets, respectively.