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Raman micro-spectroscopy and different approaches for multivariate analysis were used for an investigation of subcellular regions of X-ray exposed single SH-SY5Y human neuroblastoma cells. Nucleus and cytoplasm regions of single cells were investigated after X-rays irradiation (0, 2, 4, 6 and 8 Gy). Cells fixed immediately after irradiation and 24h irradiation were considered. Principal component analysis (PCA) and interval-PCA (i-PCA) were used for analyzing the spectra in order to highlight the changes due to the different treatments. Biochemical changes occurring in the nucleus and cytoplasm regions of single cells upon X-ray irradiation were observed. The analysis of Raman spectra allowed us to detect modifications in the contribution from proteins, nucleic acids, lipids, and carbohydrates of cells, induced at different extent on the two cell regions. The biochemical changes occurring in these cells were also discussed by using an alternative approach, namely the analysis of difference spectra, obtained by subtracting the cytoplasm-related spectrum from the corresponding one detected at the nucleus. The proposed approach enabled us to evidence some features not outlined in previous investigations. The results showed that it is possible to study in a selective way the effects of ionizing radiation on different neuroblastoma cell spatial regions. An increase of the signal related to the nucleobases, a decrease of DNA and/or RNA backbone contribution, a protein rearrangement with changes in the secondary structure, and an increase in lipid saturation were observed. These results indicated that the development of accurate data analysis methods enabling to take into account the complexity of the Raman spectra of cells and tissues and the high number of spectra needed to consider the intrinsic variability of biological samples.

Three-dimensional (3D) polymeric scaffolds are utilized in tissue engineering to provide biomechanical support for the seeded cells until they are organized into a functional tissue. Studies of cell migration are important for understanding a variety of physiological and pathological processes in tissue regeneration. Optical techniques can be employed for monitoring cell migration processes in 3D scaffolds. In particular, two-photon microscopy (TPM) can be used for monitoring these processes for living cells also at considerable depths. In the present work, the cell migration process of alive chondrocytes labelled with vital PKH67 in 3D collagen-based scaffold has been monitored at different times from the seeding process. The results show that TPM can evidence the presence of living cells in different spatial regions whose localizations depend on the location and time elapsed from the seeding.

Laccases are cuproproteins belonging to the group of oxidoreductases that are able to catalyze the oxidation of various aromatic compounds (particularly phenols) with the concomitant reduction of oxygen to water. They are characterized by low substrate specificity and have a catalytic competence which widely varies depending on the source. Additionally, laccases have also very peculiar optical properties due to their copper active sites which participate to the reduction processes. All these characteristics make laccases very flexible biotic element for biotechnological applications. During the years, a number of studies have been devoted at exploiting catalytic properties of laccases and very few at profiting of their optical properties. Some preliminary studies by absorption, fluorescence FT-IR and Raman spectroscopies of commercial laccases have evidenced their potential usefulness for optical biosensing of phenol compounds as cathecol. Moreover the sol-gel process offers a convenient and versatile method for preparing optically transparent matrices at room temperature that can represent a very convenient support for laccase immobilization. Also for immobilised enzymes the above-mentioned techniques have allowed a detailed characterization of their optical properties that confirmed the potentials of laccases in optical biosensors and represented a fundamental step in the designing of an optimised optical biosensing scheme.

The investigation of materials suitable for enzyme immobilization in biosensing applications has a widespread interest. There are many studies on physico-chemical properties of these materials at macroscopic level but few studies have been devoted to examine and correlate these properties at microscopic level. FT-IR spectroscopy with Micro-Attenuated Total Reflection (Micro-ATR) approach can be extremely useful for understanding a variety of aspects of materials which can be used for optimising immobilization procedures. Moreover, this experimental approach is particularly simple to use (no sample preparation is required) and minimally invasive. Using a Perkin Elmer Spectrum One FT-IR spectrometer equipped with a mercury-cadmium-telluride detector and a micro-ATR element we investigated different materials used for immobilization procedures with various enzymes widely used for biosensing in environmental and clinical applications. In particular, composite membranes constituted by a chemically modified poly-acrylonitrile (PAN) membrane plus layers of tethered chitosan of different molecular weight have been examined. Also silica gel matrices without and with glucose oxidase have been investigated. Spectra have been analysed and the contribution of principal functional groups has been evidenced.

An "in vitro" study of Raman spectra from oral human tissues is reported in order to the develop a diagnostic method suitable for "in vivo" oral pathology follow-up. The investigated pathology is Pemphigus Vulgaris (PV) for which new techniques for guiding and monitoring therapy would be particularly useful. Raman spectra were obtained in the wavenumber regions from 1000 to 1800 cm -1 and 2700 to 3200 cm -1 from tissues from patients at different stages of pathology (active PV, under therapy and in PV remission stage) as confirmed by histopathological and immunofluorescence analysis. Differences in the spectra depending on tissue illness stage arise in 1150-1250 cm -1 (amide III) and 1420-1450 cm -1 (CH 3 deformation) regions and around 1650 cm -1 (amide I) and 2930 cm -1 (CH 3 symmetric stretch). A wavelet deconvolution procedure was applied to the spectra for better discriminating among the three different stages of illness and a linear regression analysis was used to fully exploit the content of information of Raman spectra.

A wavelet multi-component decomposition algorithm has been used for data analysis of micro-Raman spectra from human biological samples. In particular, measurements have been performed on some samples of oral tissue and blood serum from patients affected by pemphigus vulgaris at different stages. Pemphigus is a chronic, autoimmune, blistering disease of the skin and mucous membranes with a potentially fatal outcome. The disease is characterized histologically by intradermal blisters and immunopathologically by the finding of tissue bound and circulating immunoglobulin G (IgG) antibody directed against the cell surface of keratinocytes. More than 150 spectra were measured by means of a Raman confocal microspectrometer apparatus using the 632.8 nm line of a He-Ne laser source. A discrete wavelet transform decomposition method has been applied to the recorded Raman spectra in order to overcome related to low-level signals and the presence of noise and background components due to light scattering and fluorescence. The results indicate that appropriate data processing can contribute to enlarge the medical applications of micro-Raman spectroscopy.

Pemphigus vulgaris is a chronic, autoimmune, blistering disease of the skin and mucous membranes with a potentially fatal outcome. In this case micro-Raman spectroscopy (m-RS) can provide a powerful tool for a not invasive analysis of biological tissue for biopsy and in vivo investigation. Based on the evaluation of molecular vibration frequencies, the m-RS is able to detect the main molecular bonds of protein constituents, as the C-H and C-C ones. Changes in frequency or in the relative intensity of the vibration modes revealed by m-RS can be related to changes of chemical bond and of protein structure induced by pathology. Quantitative information on the intensity variation of specific Raman lines can be extracted by Partial Least Square (PLS) analysis. m-RS was performed on some samples of oral tissue and blood serum from informed patients affected by pemphigus vulgaris (an oral pathology) at different pathology stages. The spectra were measured by means of a Raman confocal microspectrometer apparatus using the 633 nm line of a He- Ne laser source. The main protein bonds are clearly detectable in the considered samples giving important information on the integrity and on the state of tissue and blood serum components (lipids and proteins), and consequently on the occurrence of pathology.

Nowadays laser sources are largely adopted in dentistry due to their unique properties making them good candidates to substitute traditional scalpel and conventional diamond bur in the surgery of the soft and hard oral tissue, respectively. The large use of laser sources outside the research laboratories without the need of highly specialized personnel can ask for a widespread knowledge of safety issues related to this kind of equipment. The main hazard of accidental exposures regards eyes injury but increasing the power of the laser beam also skin can be involved. Safety legislations in Europe and U.S.A. take into account non ionizing radiations and laser radiation for the hazards for the health deriving from physical agents. Laser safety standards introduce 3 useful parameters for hazard characterization: "Accessible Emission Limit" (AEL), "Maximum Permissible Exposure" (MPE) and "Nominal Ocular Hazard Distance" (NOHD). We measured the MPE and NOHD for Er:YAG and other laser sources currently adopted in dentistry and we compared our results with data elaborated from standards in order to single out safe and comfortable working conditions. In fact an experimental assessment of the hazard parameters and the comparison with those of reference from safety standards turns out to be useful in order to estimate the residual hazard that can be still present after applying all the engineering protection and administrative rules.

The formation of a hybrid layer at the dentin/resin interface is a critical point in the curative processes. New restorative materials are currently under investigation in order to improve the characteristics of this interface layer. In this paper micro-Raman spectroscopy and Environmental Scanning Electron Microscope (E-SEM) analysis have been performed to investigate the interface properties of three different restorative materials. The experimental investigation has been performed on tooth cavities prepared both by Er:YAG laser ablation and by conventional diamond bur. Laser prepared cavities were realized using 100 ms pulsed light beam with energy of 350 mJ, at a frequency of 20 Hz. The cavities were filled with three different composites: True-Vitality (Den-Mat), Enamel Plus (Micerium), Supreme (3M). The treated samples were sectioned perpendicularly to the exposed dentin surface and the morphological characterization of the samples was performed by means of E-SEM operating in wet mode. This equipment permits to obtain a high-resolution image of surface without conductive coating process. The same samples were then examined by micro-Raman spectroscopy that has already shown its validity in the study of dentin/resin interfaces.

Results on image quality parameters of a time-resolved imaging system are reported. Spatial resolution has been investigated by using the edge response function method (ERF) whereas a small absorbing inclusion hidden inside a turbid slab has been adopted to analyse the contrast and noise of images obtained by different reconstruction algorithms.

Cavities in human teeth were fabricated by means of Er:YAG laser and compared with samples processed by using conventional drill. The cavities of lased and unlased samples were cured with two different composite resins ( Bis-GMA vs PEX) to investigate differences in their adhesion properties. Morphological characterization of the sample surfaces by SEM analysis was performed. The same samples were then examined by micro-Raman spectroscopy by using He-Ne laser visible light excitation, in order to characterize the dentin/resin interfaces. The Raman spectra exhibited typical peaks of dentine. Resin contributions to the Raman spectra were also detected and have been used to monitor the interface properties of the boundary region. The dependence of the Raman spectrum on spatial position has been monitored. In both lased and unlased samples the PEX diffusion in dentine is limited in a region of order of 10 μm depth. A larger interdiffusion region is observed at the boundary of dentine and Bis-GMA resin.

The possibility of obtaining images of human organs by optical techniques for diagnostic purposes is still under intense investigation. Useful information for devising the most efficient data acquisition procedures and reconstruction algorithms can be obtained by imaging well-controlled small inhomogeneities using different reconstruction procedures. For this purpose we performed time-resolved imaging experiments by embedding in a turbid slab small inclusions with different absorption coefficients. The ability of the imaging system to detect these hidden objects strongly depends on its optical properties and on the method that is adopted to reconstruct images. To this regard, in order to construct images different procedures can be adopted. The efficiency of these different procedures has been valuated by investigating the contrast, the noise and the effective signal-to-noise ratio of constructed images. Furthermore, this data analysis has been used to study the role of the inclusion’s object contrast on the efficiency of the adopted reconstruction algorithm.

Fluorescence spectra of scattering and non scattering solutions of rhodamine 6G and IR125 have been measured. The experimental data have been analyzed with a Gaussian parametric model to give some parameters that are related to the changes of scattering properties of fluorescent solutions. In addition, complementary measurements of diffuse reflectance have allowed us to test an analytical model generally used in order to recover intrinsic fluorescence spectra.

A method based on the control of surface temperature has been successfully applied to laser hardening of steels. In this way, a very simple approximation is used to predict the hardened depth and deeper treatments are obtained. In addition, this apparatus has allowed a complete characterization of the coatings generally used, thus making the heat treating process independent of their properties. Hence, performing heat treatments at a high automation level is possible.