## 4 Rationale of Proton Beam Therapy
The DU-145 cells were placed on Mylar foil, as were the PC3-cells, approximately 16-18 h before the irradiation treatment and were irradiated with the counted number of protons (50, 200, 400, 2000, and 4000 protons per cell). In addition, in this case, cells were fixed in 70% ethanol at 4 \({}^{\circ}\)C.  
Spectra were collected using the two abovementioned uFTIR spectroscopic setups. All measurements were performed at the SISSI beamline, ELETTRA Laboratory, Trieste, Italy. A Bruker Vertex 70 coupled with the Vis/IR HYPERION 3000 microscope. The SR-FTIR spectra were acquired in the transmission mode with a spectral resolution of 4 cm\({}^{-1}\) in the 4000-600 cm\({}^{-1}\) wavenumber region. For each spectrum, 512 scans were co-added to reach a good signal-to-noise ratio. An aperture of 12 \(\times\) 12 \(\upmu\)m was used for collecting single-cell spectra. The FTIR images were also obtained using a Gobar and FPA detector with 15\(\times\) Cassegrain objective in the transmission mode in the 4000-950 cm\({}^{-1}\) wavenumber region. For each spectrum, 64 scans were added with the same spectral resolution of SR-FTIR. In Reference [54], the authors focused their attention on the spectral ranges related to the contributions of various cell components. Preliminarily, they examined the phosphodiester symmetric stretching region (i.e., 1130-900 cm\({}^{-1}\)) and the CH\({}_{2}\)/CH\({}_{3}\) stretching spectral range (i.e., 3000-2800 cm\({}^{-1}\)) using both FTIR apparatus. The two apparatus gave similar results, but the absorption of the Mylar foil on which the cells were seeded altered the spectral trend in the phosphodiester asymmetric stretching region. A dose dependence was observed for all the spectra as evidenced by the second derivatives' approach (see Figures 2 and 3 in Reference [54]). Changes in DNA were evidenced at 970 cm\({}^{-1}\) due to the deoxyribose-phosphate skeletal motions (C-C) especially for the 2000 and 4000 proton doses. The authors attributed this effect to SSB, DSB, crosslinks, and deoxyribose damage in agreement with References [53; 55]. Differences were also present for the O-P-O symmetric stretching mode at 1090-1080 cm\({}^{-1}\) and the C-O furanose stretching vibrations at 1020 and 1060 cm\({}^{-1}\). Since furanose is a relevant biomarker for base-pairing and base-stacking in RNA, the authors suggest that changes in RNA molecules due to the proton exposure could cause the occurrence of improper translation processes. The analysis of the 3000-2800 cm\({}^{-1}\) spectral region, in which CH\({}_{2}\) and CH\({}_{3}\) symmetric and asymmetric stretching modes features were present, indicated changes in intensity that can be related to phospholipid membrane compaction following peroxidation [54]. The phosphodiester (i.e., 1130-950 cm\({}^{-1}\)) and the phospholipid (i.e., 3000-2800 cm\({}^{-1}\)) regions were also examined by using PCA of the second derivatives. The resulting scores and loading plots for the SR-FTIR and Gobar IR source were quite similar. This analysis indicated that the modifications induced in the cellular spectra by proton irradiation were reproducible, and for both experimental approaches, PCA could individuate six cell groups (i.e., the control and the five irradiated groups of cells). In Reference [54], a detailed description of the results of the PCA can be found. The most relevant result is related to the dose-dependent changes in the relative intensities of the DNA peak at 970 cm\({}^{-1}\) and the shift of O-P-O that can be correlated to DNA damage (single- or double-stranded breaks). The effects of proton exposure were also investigated in A-172 glioblastoma cells [56]. In this study, the authors focused their attention on the spectral changes occurring in single cellular nuclei isolated from cells treated with protons. In this paper, AFM-IR (atomic force microscopy-IR) measurements were also performed for taking advantage of the increased spatial resolution offered by this approach.