## 4 Rationale of Proton Beam Therapy
A criticality is represented by the fact that most of the existing data focus on the radiobiological effects only in the SOBP region, and therefore in the tumour area, while there is still relatively little knowledge on the sub-lethal effects on healthy tissue related to the remaining areas of the Bragg profile, such as the plateau region and the region immediately behind the SOBP, with the second being of particular relevance for carbon ion hadron therapy due to beam fragmentation process occurring after the passage in the tumoral region [38]. The dose released to healthy tissues is a source of sub-lethal damage that, if poorly repaired and inherited by the progeny, can lead to the formation of chromosomal alterations which are, in turn, potentially carcinogenic [36; 39; 40; 41]. More detailed biophysical models on the effects of high LET radiation along the entire widened Bragg curve are therefore necessary. Figure 4: Distribution of dose percentage in respect of penetration depth for a spread-out Bragg peak of a particle beam (red line). The SOBP dose distribution is the result of the convolution of different mono-energetic Bragg peaks (blue lines). The depth–dose distributions of a 10 MV photon beam (black line) is reported for confrontation. The few studies conducted for this purpose show that there is a relevant change in the radiation-induced damage along the Bragg curve, depending on the choice of the ion, on the dose and the examined endpoint (aberrations, micronuclei, senescence, etc.) [43]. As said before, it is decisive to know what biological effects are induced, in the region behind the SOBP, especially for heavy ions, for which a \"tail\" in the dose delivered exists, which is due to the fragmentation of the projectiles in the incident beam. Secondary ions with a lower Z value and less energy (hence with a higher LET) will be produced, and they will stop in the healthy tissue, contributing to the total dose released from the primary beam itself. The Contribution of the SR-\(\upmu\)FTIR Spectroscopy to the Characterization of Proton Irradiated Cells  
The enormous potentialities of SR-\(\upmu\)FTIR spectroscopy in examining cell samples exposed to different external agents have been largely exploited in various experimental conditions [6; 7; 8; 9; 10; 11; 12]. As said before, a limited number of researchers has adopted SR-\(\upmu\)FTIR spectroscopy for characterizing the effects of proton beam exposure on different human cell lines. A preliminary characterization of proton-induced effects by means of \(\upmu\)FTIR spectroscopy was reported for single PC-3 cells using a commercial system with a conventional infrared source [44]. For proton treatment, cells were seeded on 35 mm diameter Petri dishes with 10 mm round holes. The bottom of the Petri dish was capped with a 1.5 \(\upmu\)m thick Mylar foil. About 10,000 cells in 10 \(\upmu\)L medium were seeded in the central part of the Mylar foil 16-18 h before the experiments. When cells had adhered to the foil (4 h after the seeding), 2 mL of medium was added. A 2 MeV focused proton microbeam extracted from the Van de Graaff accelerator was used for irradiating cells with four doses of protons in the range of 1000-8000 protons per cell and next incubated with the medium at 37 \({}^{\circ}\)C and an atmosphere of 5% CO\({}_{2}\) for up to 24 h. After, all cells were washed with PBS and fixed in 70% ethanol at 4 \({}^{\circ}\)C. The experimental control group consisted of non-irradiated cells, processed in the same way. Spectra were collected in transmission mode with a resolution of 4 cm\({}^{-1}\), in the spectral range of 600-4000 cm\({}^{-1}\) for all groups of cells. After pre-processing analysis (see Reference [45] for details), the spectra were analyzed using the abovementioned deconvolution procedure by fitting the spectra with a sum of Gaussian-Lorentzian curves (see Figure 2 of Reference [44]).