Maria Lasalvia

Scale-independent roughness value of cell membranes studied by means of AFM technique.

The roughness of cell membrane is a very interesting indicator of cells 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.

Vibrational Characterization of Synthetic Eumelanin by Means of Raman and Surface Enhanced Raman Scattering

Eumelanin biopolymer synthetized from L-DOPA has been characterized by Raman and Surface Enhanced Raman Spectroscopy (SERS). Although Raman spectra of eumelanin were scarcely resolved, SERS measurements of this biopolymer on nanostructured gold substrate, provided a large enhancement of vibrational peaks related to functional groups adherent to the substrate, so that well resolved vibrational spectra were obtained. The SERS peaks correspond to vibrational modes related to the monomeric units of eumelanin. So, SERS spectra can be used to identify the monomeric content of eumelanin and resolve the presence of vibrational modes which have been calculated and predicted according to different theoretical models and previously observed in vibrational spectra of single monomeric units.

Visible micro-Raman spectroscopy of single human mammary epithelial cells exposed to x-ray radiation.

Abstract. 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.

Detection of pesticide effects in human keratinocytes by means of Raman microspectroscopy

Biochemical modifications of single cells of human keratinocytes exposed to different concentrations of chlorpyriphos solutions were detected by means of Raman microspectroscopy. The modifications mainly consist of breakdown of amide linkage between aminoacids. Fragmentation of DNA bases and single aminoacids occurs as well. The protein and DNA damage are already detectable at a chlorpyriphos concentration value (10−6M), which is at least three orders of magnitude lower than the concentration estimated as cytotoxic (10−3M). Raman microspectroscopy technique results to be a very sensitive technique to detect molecular changes even in single human cells exposed to toxic pesticide.

Raman Spectroscopy of Human Neuronal and Epidermal Cells Exposed to an Insecticide Mixture of Chlorpyrifos and Deltamethrin

Many pesticides are increasingly used in combinations for crop protection. Their chemical stability ensures the presence of such mixtures, both in the workspaces of the operators involved in agricultural activities and in foodstuffs, thus making probable human exposure to such chemicals in the environment. This investigation, performed by means of Raman microspectroscopy and principal component analysis, concerns the effects of in vitro cellular exposure to a commercial insecticide based on a chlorpyrifos and deltamethrin mixture. The investigated cells belong to the SHSY-5Y and human keratinocyte (HUKE) cell lines, which can be considered representative of neuronal and epidermal cells, respectively. After 24 h exposure at a concentration one-tenth of that usually used by operators, about 50% of the investigated cells were dead and the relative content of the biochemical components of both types of cells that were still alive had been affected by the exposure. A statistically significant decrease in the protein and nucleic acid content occurred in the SHSY-5Y cells, and a lowering of the lipid and carbohydrate content was observed in the HUKE cells. This study shows the utility of Raman microspectroscopy and principal component analysis for the investigation of the effects on human cells of environmental exposure to any chemicals.

Atomic force microscopy investigation of morphological changes in living keratinocytes treated with HgCl 2 at not cytotoxic doses

Morphological changes of normal human keratinocyte cells have been monitored by means of atomic force microscopy after the exposure at a mercury solution containing HgCl2 at 10−7 M. The measurements have been carried out in contact mode in a thermostated liquid cell, to reproduce a cellular environment similar to the physiologic one. Remarkable alterations of the cellular morphology and volume have been revealed after few minutes from starting the exposure experiment, although the HgCl2 concentration is several orders of magnitudes lower than the cytotoxic value (10−4 M). The atomic force microscopy technique results to be a powerful mean to investigate modifications induced in the cell morphology by external chemical agents.

Atomic force microscopy study on human keratinocytes treated with HgCl2

Morphological modifications of the cellular membrane of human keratinocytes treated with HgCl2 at different concentrations were investigated employing atomic force microscopy and Raman microspectroscopy techniques. Important changes in the surface structure of the keratinocytes membrane occur when this chemical treatment is performed. Mercury action gives rise to roughness and hole-like depressions, especially at cytotoxic concentration. Such surface features are mainly localized in peripheral zones of cells. Although the viability assay reveals that the exposure of keratinocytes to HgCl2 at a concentration of 10−6 M has no cytotoxic effect, morphological modifications are also evident in cellular membrane at such concentration. Raman microspectroscopy measurements suggest that such morphological modifications are related to modifications occurring in the lipidic layer. Our findings show that atomic force microscopy can be a valid and useful tool in studying the changes in morphology of cells when exposed to chemical stress.

Ultrafast transient absorption of eumelanin suspensions:the role of inverse Raman scattering

An ultrafast investigation is carried out on synthetic eumelanin suspended either in water or in DMSO-methanol. Upon photoexcitation by visible femtosecond pulses, the transient absorption (TA) dynamics of the suspensions are probed in a broad visible spectral range, showing clear nonlinearities. The latter arise from pump-probe interactions that induce the inverse Raman scattering (IRS) effect. We show how eumelanin TA dynamics are modified in proximity of the solvent Stokes and anti-Stokes scattering peaks, demonstrating that IRS affects the sign of TA but not the relaxation times. We compare the results obtained in both suspensions, unveiling the role of the surrounding environment. Eventually, the intrinsic response of synthetic eumelanin to ultrafast photoexcitation is evaluated.

Micro-Raman spectroscopy on human mammary epithelial cells irradiated by different doses of X-Rays

A Micro-Raman spectroscopic investigation has been performed on human mammary epithelial cells irradiated with different doses of X-ray. Results show that this experimental approach can detect radiation-induced changes in structure, protein, nucleic acid, lipid, and carbohydrate content. Micro- Raman spectroscopy appears to be a very sensitive technique to detect molecular changes even in single human cells following exposure to ionising radiation.