Aims: Until now, studies related to the morphometric and morphological variations produced on peripheral blood leukocytes from healthy donors by exposure to 1.8 GHz electromagnetic radiation (EMR) yielded controversial results. The aim of this research work is to increase the statistics regarding the above mentioned variations. Methodology: By using a reverberation chamber, which provides a controlled EMR intensity, 108 samples of human leukocytes from healthy donors were exposed to EMR of different intensities (12±4 V/m, 22±6 V/m, 42±9 V/m and 78±10 V/m) for times ranging from 5 min to 24 h. Sham exposed blood samples of the same donors were considered as controls. Using a computerized morphometric method, microscopic observations of the area size occupied by each cell were conducted; in each case the dimensional measurements were carried out on three different samples (from different donors). Besides, morphological observations were conducted staining smeared blood samples with May-Grünwald-Giemsa. Results: Exposed and sham exposed leukocytes average size was compared using the Statistical GraphPad Prism 5.0 software. In 18% out of 108 cases examined, no effects dependent on EMR have been revealed. On the contrary, statistically significant variations in area of exposed leukocytes in comparison to non exposed cells were observed in 82% out of 108 cases examined. In 68% out of 108 cases an increase in leukocyte areas was demonstrated along with morphological variations of cells; in 14% out of 108 the cases, a decrease in leukocyte areas was observed. Conclusions: Even though this study needs a functional evaluation of leukocytes exposed to EMR, our results suggest that 1.8 GHz EMR is able to produce increase in the leukocyte areas as well as morphological alterations.

Bright field microscopy and atomic force microscopy techniques are used to investigate morphological properties of synthetic eumelanin, obtained by oxidation of l-DOPA solution, deposited on glass andmica substrates. Deposits of eumelanin are characterized by aggregates with different shape and size. On a micrometric scale, filamentous as well as granular structures are present on glass and mica substrates,with a larger density on the former than on the latter. On a nanometric scale, filamentous aggregates,several microns long and about 100 nm wide and high, and granular aggregates, ∼50 nm high and 100 nmwide, are found on both substrates, whereas point-like deposits less than 10 nm high and less than 50 nmwide are found on mica substrate. Dynamic light scattering measurements and atomic force microscopy images support the evidence that eumelanin presents only nanometric point-like aggregates in aqueous solution, whereas such nanoaggregates organize themselves according to granular and filamentousstructures when deposition occurs, as a consequence of interactions with the substrate surface.

Optical properties of synthetic and natural eumelanin are presented and compared, in order to investigate the structural organization of eumelanin, which is related to the function of this biopolymer. Synthetic eumelanin is produced by oxidation of tyrosine with hydrogen peroxide, whereas natural eumelanin is extracted from Sepia Officinalis and from Rana Esculenta. Vibrational spectroscopy techniques (as Raman scattering and infrared absorption) show that both types of biopolymer include chemical functional groups characteristic of the monomeric units of eumelanin, although natural eumelanin includes also protein-related groups, proportionally to the protein content. X-ray diffraction spectra are in agreement with the hypothesis that eumelanin monomers assembly themselves and form protomolecules consisting of stacked layers (distant 3 – 4 Å each other) of indolic sheets. Absorption measurements, characterized by a monotonic increase of optical density from near-IR to UV range, support the model that eumelanin consists of a distribution of aggregates of oligomeric structures having different size and chemical composition. The estimated values of the optical gap indicate that the natural eumelanins are characterized by a larger structural disorder than the synthetic one. Fluorescence spectra confirm that the biopolymer consists of ensembles of chemically distinct oligomer systems, which can be selectively excited. This result is also supported by Dynamic Light Scattering measurements, which permit to visualize the distribution of particles size. In fact, the nanoaggregate systems of natural eumelanin have a larger size than those of synthetic eumelanin. This might be related to the biological functions of such a biopolymer, particularly as far as photoprotective action is concerned.

Raman spectroscopy is a useful technique for early diagnosis of cellular damage related to the exposure at toxic chemicals, because biochemical changes related to action mechanism of chemicals can be detected in Raman spectra. In this investigation Raman microspectroscopy has been used, in correlation with the principal component analysis method, to detect biochemical changes occurring in cultured human cells as a consequence of exposure at a commercial pesticide. Cultured human keratinocyte cells have been exposed at increasing concentrations of pesticide for 24 h. Viability tests indicated that the cells vitality is almost completely preserved when the concentration of active ingredient of pesticide is very low (5 108 M, about two orders of magnitude lower than the cytotoxic concentration at 24 h exposure). Nonetheless, the analysis of Raman spectra allows to state that a biochemical change occurs: it involves mainly the protein linkages between aminoacids (amide I bonds) and, at a minor amount, lipids. On the whole, principal components analysis is able to classify into two separate clusters the control and exposed human cells. 2011 Elsevier B.V. All rights reserved

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.

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. © 2012 Elsevier B.V. All rights reserved

In Ar-rich Ar–H2–CH4 gas mixture the presence of H2 is found to be beneficial to the plasma stability. On the other hand, too high H2 percentages lead to materials showing a high surface roughness. In the present work, diamond films were grown on p-type Si (100) substrates screening different quantities of H2. The plasma phase and plasma–substrate interface were investigated by in-situ optical emission spectroscopy and pyrometric interferometry to determine the behavior of emitting species and the deposition rates, respectively. The obtained films were characterized by Raman micro-spectroscopy, AFM and SEM techniques. For H2 percentages between 6.3 and 10%, the structure and morphology are characteristic of nanocrystalline films, affording low roughness values when a buffer layer was grown between the diamond coating and the treated silicon surface.

Pesticides are chemical compounds widely used in agricultural and industrial activities, as well as domestic ones. A commercial pesticide is usually composed of active ingredients and formulants. Among the active ingredients, Deltamethrin is a pyrethroid chemical widely used for synthesizing pesticides products which are very effective in damaging the central nervous system of pests. In this work, we analyze, by means of atomic force microscopy (AFM), cellular morphological changes induced by exposure to a Deltamethrin-based commercial pesticide (Decaflow). AFM microscopy, in addition to the well-known characterization of the cellular topography, has the ability to monitor interesting biomechanical parameters of the surface as roughness and elastic modulus. In particular, we exposed normal human keratinocytes for 24 hours at different solutions of Decaflow, well below the threshold of cytotoxicity. The AFM images of exposed cells show alterations of surface cell shape. Moreover exposed cells are characterized by an increase of the value of membrane roughness. The mechanical properties of cells are also modified after Decaflow exposure, as confirmed by a decrease of the elasticity modulus with increasing the concentration of pesticide.

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.

Micro-Raman technique can be particularly useful to investigate the chemical changes induced in structure, protein, nucleic acid, lipid, and carbohydrate contents of cells. The aim of this work is to inspect the possibility to employ Raman microspectroscopy to detect biochemical modifications in human mammary epithelial cells after exposure to different Xray doses. The samples consisted of cells cultured on polylysine-coated glass coverslips. After the exposition, control and treated cells were washed in phosphate-buffered saline (PBS) and then fixed in paraformaldehyde 3.7%. They were examined using a confocal micro-Raman system equipped with a He-Ne laser (λ = 632.8 nm; power on the sample= 3.5mW). Differences in the intensity ratio of specific Raman vibrational markers commonly assigned to phenylalanine and tyrosine amino acids (at 1000, 1030, 1618 cm-1), DNA bases (787, 1090, 1305 cm-1), and amide III (1237, and 1265 cm-1) with respect a reference peak (the one of lipids at 1450 cm-1) were evidenced between control and exposed cells. These differences may be indicative of damage in exposed cells as the fragmentation of individual amino acids and DNA bases, crosslink effects in molecular structure of DNA and protein conformational change that especially tend to "unwind" the protein due to the breaking of hydrogen bonds between peptide chains.