## 4 Conclusions  
Our results concerning MCF-10A cells fixed immediately after the end of the exposure to radiation suggest that the use of MirrIR substrate can offer a fast, reliable, and less expensive experimental approach for proton irradiation and \(\upmu\)-FT-IR investigation, considering the greater ease of handling of samples during preparation and irradiation phase (e.g., low risk of breakage compared to thin mylar sheets). This can be particularly useful when \(\upmu\)-FT-IR spectroscopy is performed for investigating large area samples in which many cells are positioned, as in the present case. The spectra obtained for normal human breast cells exposed to different doses of protons and fixed immediately after the irradiation show spectral characteristics similar to those obtained with other experimental approaches previously mentioned [12; 13; 14].  
The spectra analysis performed by using deconvolution procedures and the evaluation of the ratio between the area of selected peaks allowed us to prove changes in the DNA and lipid regions, in agreement with the results previously reported [12; 14; 42]. The Amide I and Amide III regions were also studied and the occurrence of faint changes in protein secondary structure due to proton irradiation have been evidenced for the first time to our knowledge.  
The results here reported can be considered as a starting point to evaluate the use of the proposed approach for FT-IR spectroscopy in the field of cells radiobiological response. However, other steps are needed before such a technique is established as a predictor of cellular radio response. In fact, our results pertain to cells fixed immediately after radiation exposure, but it is known that for several hours after irradiation cells try to repair radiation-induced DNA damage. Therefore, future measurements should be performed using cells fixed at different times after exposure, in order to evaluate the evolution over time of the biochemical alterations produced by proton irradiation and possibly compare them with those elicited by photon irradiation as used in conventional radiotherapy.  
**Author Contributions:** Conceptualization, M.L. ([PERSON]) and V.C.; methodology, G.P. ([PERSON]), M.L. ([PERSON]), M.P., F.C., P.P.; software, V.R., I.D.; formal analysis, V.R., M.P.; investigation, M.P., V.R., M.L. ([PERSON]), F.P.C., P.P., L.M., G.P. ([PERSON]); data curation, G.P. ([PERSON]), V.R., M.P., LD.; writing-original draft preparation, M.L. ([PERSON]); writing-review and editing, M.L. ([PERSON]), V.C.; funding acquisition, V.C., L.M. All authors have read and agreed to the published version of the manuscript.  
**Funding:** The financial support of this research was provided by INFN (Istituto Nazionale di Fisica Nucleare), through the research project ETHICS (\"Pre-clinical experimental and theoretical studies to improve treatment and protection by charged particles\").  
**Institutional Review Board Statement:** Not applicable.  
**Informed Consent Statement:** Not applicable.  
**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.  
**Acknowledgments:** The authors are pleased to thank [PERSON] for his valuable help and discussion.  
**Conflicts of Interest:** The authors declare no conflict of interest.