ABSTRACT: In a previous study, an apparatus generating 1.8 GHz electromagnetic radiation for “in vivo” biomedical study was designed and implemented. The apparatus consisted of a reverberation chamber and it reproduced a habitat similar to the usual one for the laboratory animals. Plexiglas boxes with 300 cc physiological liquid were utilized as simple phantoms. For the measurements a small Electric Field Probe was used. The maximum SAR (Specific Absorption Rate) and average Power Efficiency Pe (SAR/input power) values obtained were quite low (“in vivo experiments”) and this was the drawback of the apparatus. In the present work, different modifications introduced in order to increase SAR and Power Efficiency are presented. In the new configuration, the dosimetry for the previous phantoms and for oil-in-gelatine phantoms was investigated and quite satisfactory SAR and Power Efficiency values were obtained, overcoming the previous drawback. In the first case the sensor was waterproofed, as in the previous study; in the other case a Plexiglas box with inside a tight shaped allocation for the measurement probe was realized. These measurement technologies could be applied to other media used for the phantoms.

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.

Raman spectroscopy is a powerful technique for studying cellular biochemistry. In fact, each toxic chemical induces biochemical changes related to the own action mechanism. In this investigation Raman microspectroscopy has been used, in correlation with atomic force microscopy images, to detect biochemical and structural damages occurring in culturedhumancells as a consequence of deltamethrin exposure. Cultured human keratinocyte cells have been exposed at increasing concentrations of deltamethrin from 10−3M to 10−6M for 24 h. A viability test indicated that the cytotoxic dose corresponds to exposure at deltamethrin solution for 24 h with the chemical concentration between 10−4Mand 2.5 10−4 M. The compared analysis of Raman spectra and AFM images allows to state that an evident damage occurs in the plasmaticmembraneand it is already detectable after exposure of keratinocytes at the lowest investigated deltamethrin concentration (10−6 M). The most important modifications are related to the breakdown of CH2 bonds of lipidic chains, whereas proteineous bonds are less involved in the deltamethrin action. On the whole, cellular damage starts after exposure to deltamethrin doses well lower than that established as cytotoxic. © 2011 Elsevier B.V. All rights reserved

Oral squamous cell carcinoma is a widespread cancer disease whose survival rate is strongly dependent on early diagnosis and on the degree of malignancy. The conventional histopathology methods, which are currently the standard ones for diagnosis, are very invasive so that they can be hardly proposed as screening methods for an early and accurate detection of disease. Raman microspectroscopy and atomic force microscopy can be potentially considered as useful tools for cancer diagnosis and detection of the malignancy degree because they provide information about the biochemical cellular content and nanomechanical properties, respectively, which would be modified by the onset and progression of pathology. The present work shows that both techniques can successfully discriminate the two cellular types of cells characterized by different degrees of oral squamous cell carcinoma. The discrimination by Raman microspectroscopy occurs according to a larger content of nucleic acids and a minor content of protein components in cells characterized by a larger degree of disease, whereas the discrimination by atomic force microscopy is achieved because of a decrease of stiffness as the degree of disease increases. Overall, both techniques could provide useful diagnostic information related to the degree of malignancy of the oral squamous cell carcinoma disease.

The deposition of as-received nanodiamond (ND) particles on silicon substrate was performed by the pulsed spray technique, using a dispersion of 250 nm ND in 1, 2-dichloroethane. A set of samples was sprayed by varying the number of pulses from 1 to 500. The morphology of the samples was characterized and monitored by means of optical, atomic force, and confocal microscopies. At a low number of pulses, sparse diamond particles were observed, whereas at a high number of pulses dense/quasi-continuous ND layers were formed. The electrical conductivity measurements of surface silicon substrate evidenced a remarkable change for the presence of ND particles. This behavior is also found by theoretical simulations (finite element method). Finally, a comparison between the electrical resistances measured on these samples versus the pulse number and the inverse current density calculated as a function of the number of ND particles, showed a good agreement. The experimental results highlighted an increase of the electrical current by using a number of pulses <100, whereas the simulation results proved the enhancement of current density and itssurface rectification by employing a specific number of particles. The current increased by increasing the temperature and during the heating–cooling cycles hysteresis was observed.

Blood is a fluid connective tissue of human body, where it plays vital functions for the nutrition, defense and well-being of the organism. When circulating in peripheral districts, it is exposed to some physical stresses coming from outside the human body, as electromagnetic fields (EMFs) which can cross the skin. Such fields may interact with biomolecules possibly inducing non thermal-mediated biological effects at the cellular level. In this study, the occurrence of biochemical/biological modifications in human peripheral blood lympho-monocytes exposed in a reverberation chamber for times ranging from 1 to 20 h to EMFs at 1.8 GHz frequency and 200 V/m electric field strength was investigated. Morphological analysis of adherent cells unveiled, in some of these, appearance of an enlarged and deformed shape after EMFs exposure. Raman spectra of the nuclear compartment of cells exposed to EMFs revealed the onset of biochemical modifications, mainly consisting in the reduction of the DNA backbone-linked vibrational modes. Respirometric measurements of mitochondrial activity in intact lympho-monocytes resulted in increase of the resting oxygen consumption rate after 20 h of exposure, which was coupled to a significant increase of the FoF1-ATP synthase-related oxygen consumption. Notably, at lower time-intervals of EMFs exposure (i.e. 5 and 12 h) a large increase of the proton leak-related respiration was observed which, however, recovered at control levels after 20 h exposure. Confocal microscopy analysis of the mitochondrial membrane potential supported the respiratory activities whereas no significant variations in the mitochondrial mass/morphology was observed in EMFs-exposed lympho-monocytes. Finally, altered redox homeostasis was shown in EMFs-exposed lympho-monocytes, which progressed differently in nucleated cellular subsets. This results suggest the occurrence of adaptive mechanisms put in action, likely via redox signaling, to compensate for early impairments of the oxidative phosphorylation system caused by exposure to EMFs. Overall the data presented warn for health safety of people involved in long-term exposure to electromagnetic fields, although further studies are required to pinpoint the leukocyte cellular subset(s) selectively targeted by the EMFs action and the mechanisms by which it is achieved.

Resultsaboutfluorescencespectroscopyofnaturaleumelaninarepresentedandcomparedto analogousresultsrelatedtosyntheticeumelanin,withtheaimtoinvestigatethestructuralorganiza- tion ofeumelaninandtheroleoftheproteincoatinitsopticalandstructuralproperties.Thespectraof syntheticandnaturaleumelaninhavesimilaropticalspectra,exceptforthefeaturesrelatedtoproteins in naturaleumelanins.Fluorescenceduetoensemblesoflargeoligomersystemshasbeenspectrally distinguishedfromthatduetomonomersandsmalloligomersystems.Nonetheless,thenatural (protein-containing)eumelaninresultstoconsistofaggregateshavingalargersizethanthatof syntheticeumelanin. & 2011ElsevierB.V.Allrightsreserved

Raman micro-spectroscopy can be used to investigate biological single cells exposed to different chemicals. Since chronic exposure at low doses of pesticides can promote several diseases, the investigation of cellular changes induced by exposure to non-cytotoxic doses of pesticides is of increasing interest. The efficiency of Raman micro-spectroscopy to detect chemical modification in normal human keratinocytes induced by exposure to non-cytotoxic doses of chlorpyriphos, an organophosphate pesticide present in many plant-protection products, was investigated. Such modification affects mainly proteineous components (both single amino acids and amide linkages between amino acids) of the nucleus, cellularmembranes and cytoplasm as well as the nucleic acid component of the nucleus. Chemical modifications are already detectable after 24 h exposure of keratinocytes at a chlorpyriphos concentration of 10−6 M, which is three orders of magnitude lower than the cytotoxic concentration (10−3 M). Heavy damage to the lipid component occurs after exposure to the nearly cytotoxic concentration (10−4 M). Atomic force microscopy images of keratinocyte cells exposed for 24 h to various chlorpyriphos concentrations show a progressive deterioration of the morphology of cellular membrane as the chlorpyriphos concentration increases. The results of this work may have wide applications in the monitoring of molecular changes in single human cells exposed to toxic agents. Copyrightc 2010 JohnWiley & Sons, Ltd.

The mechanical properties of polycrystalline diamond coatings with thickness varying from 0.92 to 44.65 μm have been analysed. The tested samples have been grown on silicon substrates via microwave plasma enhanced chemical vapour deposition from highly diluted gas mixtures CH4–H2 (1% CH4 in H2). Reliable hardness and elastic modulus values have been assessed on lightly polished surface of polycrystalline diamond films. The effect of the coating thickness on mechanical, morphological and chemical-structural properties is presented and discussed. In particular, the hardness increases from a value of about 52 to 95 GPa and the elastic modulus from 438 to 768 GPa by varying the coating thickness from 0.92 to 4.85 μm, while the values closer to those of natural diamond (H=103 GPa and E=1200 GPa) are reached for thicker films (N5 μm). Additionally, the different thickness of the diamond coatings permits to select the significance of results and to highlight when the soft silicon substrate may affect the measured mechanical data. Thus, the nanoindentation experiments were made within the range from 0.65% to 10% of the film thickness by varying the maximum load from 3 to 80 mN. © 2010 Elsevier B.V. All rights reserved

Micro-Raman spectroscopy is a very promising tool for medical applications, thanks to its sensitivity to subtle changes in the chemical and structural characteristics of biological specimens. To fully exploit these promises, building a method of data analysis properly suited for the case under study is crucial. Here, a linear or univariate approach using a R2 determination coefficient is proposed for discriminating Raman spectra even with small differences. The validity of the proposed approach has been tested using Raman spectra of high purity glucose solutions collected in the 600 to 1,600 cm−1 region and also from solutions with two known solutes at different concentrations. After this validation step, the proposed analysis has been applied to Raman spectra from oral human tissues affected by Pemphigus Vulgaris (PV), a rare life-threatening autoimmune disease, for monitoring disease follow-up. Raman spectra have been obtained in the wavenumber regions from 1,050 to 1,700 cm−1 and 2,700 to 3,200 cm−1 from tissues of patients at different stages of pathology (active PV, under therapy and PV in remission stage) as confirmed by histopathological and immunofluorescence analysis. Differences in the spectra depending on tissue illness stage have been detected at 1,150–1,250 cm−1 (amide III) and 1,420–1,450 cm−1 (CH3 deformation) regions and around 1,650 cm−1 (amide I) and 2,930 cm−1 (CH3 symmetric stretch). The analysis of tissue Raman spectra by the proposed univariate method has allowed us to effectively differentiate tissues at different stages of pathology.

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.

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.

We report vibrational spectra of two types of eumelanin samples: a synthetic one, produced by oxidation of tyrosine with hydrogen peroxide, and a natural one, extracted fromSepia officinalis,with the aim of pointing out the differences between the main vibrational properties of such two pigments. Fourier transform infrared, Raman and surface- enhanced Raman scattering spectra allow the identification of several vibrational modes involving specific monomeric units. The main differences between the spectra of the two types of eumelanin are related to (i) a larger amount of carboxylic acid in the synthetic than in the natural sample; (ii) a shift of spectral position of corresponding peaks; and (iii) a contribution of residual proteins to the signals from the natural sample. Such results suggest that vibrational techniques may be helpful for non-destructively determining the composition of various eumelanin pigments, with potential applications in biomedical, electronic and cultural heritage fields.

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.