## 3 Results and Discussion
In particular, the mean PC1 score values for 20 h exposure are \(1.551\times 10^{-3}\pm 1.183\times 10^{-3}\) and \(-1.551\times 10^{-3}\pm 1.512\times 10^{-3}\) for unexposed and exposed cells, respectively. Therefore, for such exposure time we can assert that the mean score value of unexposed cells is positive, whereas that for exposed cells it is negative. A similar statement cannot be made for 12 h and 5 h exposure experiments, because the mean PC1 score values are \(5.495\times 10^{-4}\pm 9.075\times 10^{-4}\) and \(-5.644\times 10^{-4}\pm 1.083\times 10^{-3}\) for 12 h unexposed and exposed cells, respectively, and \(-3.848\times 10^{-4}\pm 1.313\times 10^{-3}\) and \(4.301\times 10^{-4}\pm 1.270\times 10^{-3}\) for 5 h unexposed and exposed cells, respectively. Such remarks are important to explain the loading plots shown in Figure 4. In particular, Figure 4 displays PC1 loading plots (red lines) compared with the difference average Raman spectra (black  
Figure 3: Score plots of the 1 st and 2 nd principal component of Raman spectra obtained by means of PCA technique. The data refer to the spectra of the unexposed (black dots) and electromagnetic fields (EMFs) exposed (red dots) for 20 (a), 12 (b) and 5 (c) hours. lines) between unexposed and exposed cells for the three investigated exposure times. In general, loading plots consist of spectra with positive and negative peaks: positive peaks correspond to an increasing Raman signal in the original spectra of the sample type having positive score values, whereas negative peaks indicate a decreasing Raman signal in the original spectra of the same sample type [32]. By comparing the PC1 plot in Figure 4a with the spectrum reported in Figure 1, the PC1 positive peaks for 20 h exposure can be mainly attributed to nucleic acid components (except the peak at 1700 cm\({}^{-1}\), which is unassigned because there is no corresponding resolved Raman feature in Figure 1), whereas the negative PC1 loading peaks correspond to protein components (except the peak at 1550 cm\({}^{-1}\), which has no corresponding Raman feature in Figure 1). Therefore, we can assess that PC1 plot confirms and better visualizes the relative decrease of DNA bonds in 20 h exposed PBLM cells with respect to the comparison of average exposed and unexposed Raman spectra shown in Figure 2a. In addition, the loadings of PC1, which in this case discriminate the nucleic acid from the protein components, are in good agreement with the difference spectrum between the average Raman signal of unexposed and exposed PBLM cells, as visible in Figure 4a. On the contrary, the correspondence of PC1 loading plot and difference spectrum is less evident for 12 h and 5 h exposed PBLM cells, as shown in Figure 4b,c, respectively. Therefore, for such exposure times the biochemical effects related to exposure are quite weak, so that the PCA technique, as discussed above, does not clearly differentiate inter-group and intra-group variability and the latter considerably contribute to score and loading values [18]. Figure 4: Loading plots of principal component 1 (PC1) (red line) and difference Raman spectra (black line) between 20 h (**a**), 12 h (**b**) and 5 h (**c**) unexposed and exposed cells. A multiplicative factor has been applied to the difference spectrum to better visualize the comparison with the corresponding loading 1 spectra. Because of the small difference between the Raman spectra of control and exposed PBLM cells, we estimated cellular effects induced by EMFs by also measuring the ROS production in the two types of cells. Indeed, it is reported that EMFs can induce ROS production, whose overproduction can lead to DNA damage [8; 33; 34; 35]. Typical fluorescence images of unexposed and 20 h exposed PBLM cells are shown in Figure 5a,b, respectively: they clearly suggest the increase of ROS production as a consequence of exposure to EMFs. The increase of DCF fluorescence intensity from exposed PBLM cells with respect that from unexposed ones is shown in Figure 5c for the three exposure times.