## 1 Introduction  
Fourier transform infrared micro-spectroscopy (\(\mu\)-FT-IR) has been largely demonstrated to be a valuable tool for investigating the changes occurring in human cells after ionizing radiation exposure. In particular, radiation-induced apoptosis in human lymphocytes was evidenced by [PERSON] and [PERSON] [1]. A DNA decreasing content and an increase in the bands of amide I and II of proteins in the apoptotic cells were inferred from the differences between the spectra from apoptotic and control lymphocytes. These results are in agreement with a possible pathway of radiation-induced apoptosis, which needs active protein synthesis triggered by the occurrence of DNA lesions. \(\upmu\)-FT-IR was demonstrated to be able to investigate the temporal evolution of the apoptosis process [2; 3], too. These results concerning early radiation- and oxidative-induced damage to nucleic acids and proteins in single human cells [3] can be evaluated by \(\upmu\)-FT-IR, and are well correlated with established radiobiology techniques. [PERSON] et al. [4] quantitatively analyzed the effects of different \(\upgamma\)-radiation doses on the IR spectra of the human keratinocytes cell line (HaCaT). They also investigated the effects at different times after irradiation. Using \(\upmu\)-FT-IR [PERSON] et al. [5] explored a different aspect of X-ray induced cellular processes. They investigated the radiation damage of biological samples as a limiting factor when high-resolution X-ray microscopy is performed. Recently, some of us reported a \(\upmu\)-FT-IR investigation on neuroblastoma cells (SH-SYSY cell line), in which the use of ratios between absorbances of appropriately chosen peaks was adopted for studying X-ray irradiation effects [6]. This approach represents a very direct way to monitor changes for specific cellular components. Modifications of membrane fluidity and changes in the contributions of the enzymes involved in DNA repair were demonstrated. In addition, the changes for ratio between the absorbance bands of CH\({}_{3}\) and CH\({}_{2}\) asymmetric stretching suggested the occurrence of an apoptotic process also for this cell line. Moreover, the ratios between protein and lipid contents showed significant differences that can be related to changes in membrane characteristics. In the last ten years, \(\upmu\)-FT-IR has been also applied to the study of biochemical alteration induced in different human cells by particle beams, with particular attention to proton beam irradiation due to the relevance of this treatment in radiotherapy for some cancer types [7; 8]. Proton therapy (PT) is not considered currently as the treatment of choice for breast cancer [9]. However, due to the favorable inverted dose-depth profile, which allows greater sparing of normal tissue and organs at risk compared to conventional radiotherapy, the use of PT is clinically justifiable to decrease the risks of radiation-induced cardiovascular disease (CVD) in breast cancer patients [10; 11]. Using synchrotron FT-IR spectroscopy, DNA damage and lipid alteration in proton-irradiated cells have been shown [12; 13]. These studies were conducted using different methods of sample preparation, irradiation, and spectra acquisition [12; 13; 14]. Cells were seeded on ad-hoc Petri dishes with the use of 1.5 \(\upmu\)m Mylar foil or 0.5 \(\upmu\)m silicon nitride (Si\({}_{3}\)N\({}_{4}\)) membranes. The latter substrate enabled in-vitro proton irradiation of cells and then spectra acquisition on the same substrate minimizing the number of lost cells treated by radiation [12]. The adopted substrates allow spectra acquisition in transmission geometry but are not easy to handle. The selection of proper substrates represents an important challenge in order to make the \(\upmu\)-FT-IR technique faster and easier to use [15; 16]. For this reason, many efforts are underway to identify the most suitable substrate for each type of application [17; 18; 19].