The roughness of cell membrane is a very interesting indicator of cell's health state. Atomic Force Microscopy allows us to investigate the roughness of cell membrane in great detail, but the obtained roughness value is scale-dependent, i.e. it strongly depends on measurement parameters, as scanning area and step size. The scale-dependence of the roughness value can be reduced by means of data filtration techniques, that are not standardized at nanometric scale, especially as far as biological data are concerned. In this work, a new method, based on the changes of values of some roughness parameter (root mean square roughness and skewness) as a function of filtration frequencies, has been implemented to optimize data filtering procedure in the calculation of cell membrane roughness. In this way, a root mean square roughness value independent of cell shape, membrane micro-irregularities and measurement parameters can be obtained. Moreover, different filtration frequencies selected with this method allow us to discriminate different surface regimes (nominal form, waviness and roughness) belonging to the raw cell profile, each one related to different features of the cell surface.

A micro-Raman spectroscopy investigation has been performed in vitro on single human mammary epithelial cells after irradiation by graded x-ray doses. The analysis by principal component analysis (PCA) and interval-PCA (i-PCA) methods has allowed us to point out the small differences in the Raman spectra induced by irradiation. This experimental approach has enabled us to delineate radiation-induced changes in protein, nucleic acid, lipid, and carbohydrate content. In particular, the dose dependence of PCA and i-PCA components has been analyzed. Our results have confirmed that micro-Raman spectroscopy coupled to properly chosen data analysis methods is a very sensitive technique to detect early molecular changes at the single-cell level following exposure to ionizing radiation. This would help in developing innovative approaches to monitor radiation cancer radiotherapy outcome so as to reduce the overall radiation dose and minimize damage to the surrounding healthy cells, both aspects being of great importance in the field of radiation therapy.

Fourier transform infrared micro-spectroscopy (μ-FT-IR) is nowadays considered a valuable tool for investigating the changes occurring in human cells after exposure to ionizing radiation. Recently, considerable attention has been devoted to the use of this optical technique in the study of cells exposed to proton beams, that are being increasingly adopted in cancer therapy. Different experimental configurations are used for proton irradiation and subsequent spectra acquisition. To facilitate the use of μ-FT-IR, it may be useful to investigate new experimental approaches capable of speeding up and simplifying the irradiation and measurements phases. Here, we propose the use of low-e-substrates slides for cell culture, allowing the irradiation and spectra acquisition in transflection mode in a fast and direct way. In recent years, there has been a wide debate about the validity of these supports, but many researchers agree that the artifacts due to the presence of the electromagnetic standing wave effects are negligible in many practical cases. We investigated human normal breast cells (MCF-10 cell line) fixed immediately after the irradiation with graded proton radiation doses (0, 0.5, 2, and 4 Gy). The spectra obtained in transflection geometry showed characteristics very similar to those present in the spectra acquired in transmission geometry and confirm the validity of the chosen approach. The analysis of spectra indicates the occurrence of significant changes in DNA and lipids components of cells. Modifications in protein secondary structure are also evidenced.

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