## 3 Results and Discussion  
To assess whether the HUKE cells can be discriminated from the SH-SYSY, we compared the FTIR spectra of the two types of cells grown on the mentioned four substrates. The comparison of the mean normalized spectra is reported in Figure 1, whereas the distributions of the normalized spectra are shown in the Supplementary Materials. Such spectra are characterized by several absorption features, which were assigned according to the published literature [20; 2]; the most prominent ones are labeled in Figure 1. In particular, the broad and intense band centered at about 3300 cm\({}^{-1}\) is mainly related to the amide A (N-H stretching mode of proteins amino acids and nucleic acids). The peaks observedat about 2956 and 2870 cm\({}^{-1}\) are assigned, respectively, to the asymmetric and symmetric stretching mode of the CH\({}_{3}\) groups of cellular proteins and lipids. The bands at 2924 and 2850 cm\({}^{-1}\) are assigned, respectively, to the asymmetric and symmetric stretching of the CH\({}_{2}\) groups of lipids. The mean spectra are quite similar each other, and they are also similar to those obtained by other authors for keratinocyte and neuronal cell lines. In particular, FTIR spectra of human keratinocyte HaCaT cell lines and oral keratinocytes isolated by tissue samples, both grown on MirrIR slides, were obtained by [PERSON] et al. [21] and by [PERSON] et al. [22], respectively. FTIR spectra of neuroblastoma cells SH-SY5Y grown on MirrIR slides were investigated by [PERSON] et al. [23]. Interestingly, a comparison among the spectra in Figure 1 for each substrate evidences that the main differences between the HUKE cell and SH-SY5Y cell spectra are related to the relative intensity of some peaks, as those centered at 2956 and 2924 cm\({}^{-1}\). Such a behavior suggests that a different relative content of protein and lipid components characterizes and discriminates a cellular type from the other one. The absorption intensity change of the lipid peak at 2920 cm\({}^{-1}\) in malignant cells with respect to normal ones was observed by [PERSON] et al. [17]; however, the results of these authors cannot be effectively compared to the results in Figure 1, because of the different cellular types and normalization methods. PCA technique was used to assess whether FTIR is able to discriminate between the two types of cells grown on the four substrates. In particular, the spectral range between 2750 and 3000 cm\({}^{-1}\), including absorption peaks related to lipid and protein components, was investigated by means of PCA. Figure 2 shows the PCA score plots for the two types of cells grown on the four substrates. It is  
Figure 1: Average normalized FTIR spectra of Human Keratinocyte (HUKE) (red lines) and SH-SY5Y (black lines) cells grown on CaF\({}_{2}\) (**a**), low-emissivity (low-e) (**b**), glass (**c**), and coverslip (**d**) substrates. clearly visible that HUKE and SH-SYSY are separated according to PC1 component, although the separation grade is different according to the substrate type. In order to estimate such a separation grade for each growth substrate, we used the difference between the average values of the distributions of PC1 values of the two types of cells. The estimated values are reported in Table 1. As evident in Table 1, the separation between HUKE and SH-SYSY cells was statistically significant for PC1 component regardless of growth substrate. Although CaF\({}_{2}\) and low-e substrates are able to provide large separation capability, HUKE and SH-SYSY can be also discriminated when grown on the glass and coverslip slides. The capability to discriminate different types of cells grown on glass and coverslip slides is interesting in view of a clinical application of FTIR for diagnostic purposes, because such slides are widely used in clinical settings for histological and cytological analyses. The possibility to discriminate different types of cells measured by FTIR spectroscopy was also demonstrated by [PERSON] et al.