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Natural dyes have been utilized since ancient times to produce colorful garments and, at the same time, each culture has developped its own traditional designs and techniques during the centuries. Hence, in order to accomplish a proper conservation program of these artefacts, it is fundamental the importance of studying the used dyes on historic textiles. Traditionally, the identification is carried out through invasive or micro-invasive techniques such as HPLC or Raman-SERS. However, a first screening using non-invasive approaches, that could tentatively identify some of the dyes in a contactless mode, is strongly reccommended. The present study focuses on the application of non-invasive spectroscopic techniques, namely FORS and hyperspectral imaging in the visible and near infrared range, for the identification of dyes on textiles. Twelve natural dyestuffs (twelve from plant, two insect dyes and a lichen) and three mordants (KAl(SO4)2, CuSO4 ? 5H2O, FeSO4 ? 7H2O) were selected and combined to create a set of wool samples whose reflectance spectra, acquired using FORS technique, were the basis for a spectral database. The same set was used to test the Specim IQ hyperspectral camera, with positive results for a non-invasive investigation on wider areas. The application of Spectral Angle Mapping (SAM) gave the possibility to map directly on the image pixel with similar spectral features. Finally, colorimetric data were acquired with a spectrocolorimeter to obtain chromaticity coordinates.

Laser safety glasses block visibility of the laser light. This is a big nuisance when a clear view of the beam path is required. A headset made up of a smartphone and a viewer can overcome this problem. The user looks at the image of the real world on the cellphone display, captured by its rear camera. An unimpeded and safe sight of the laser beam is then achieved. If the infrared blocking filter of the smartphone camera is removed, the spectral sensitivity of the CMOS image sensor extends in the near infrared region up to 1100 nm. This substantial improvement widens the usability of the device to many laser systems for industrial and medical applications, which are located in this spectral region. The paper describes this modification of a phone camera to extend its sensitivity beyond the visible and make a true augmented reality laser viewer.

A mechanical model of light propagation helps to show that optical refraction is related to the speed of light and how it changes from one medium to another. A tricycle toy robot is used to realize the model. Left and right wheels independently change their speed (high or low) in response to the local color (white or black, respectively) of the ground pattern detected by the corresponding optical sensor. Because of this simple rule the robot path deviates when passing, for example, from a clear to a dark area, in this way mimicking the refraction of a beam of light at an air–glass interface. Black silhouettes, representing optical components, are positioned on a clear mat along the robot trip. The robot trajectories reproduce phenomena such as refraction and total internal reflection showing a perfect analogy to light path described by geometrical optics in a sort of live ray-tracing.

Alterations of the tightly interwoven neuron/astrocyte interactions are frequent traits of aging, but also favor neurodegenerative diseases, such as Alzheimer disease (AD). These alterations reflect impairments of the innate responses to inflammation-related processes, such as β-amyloid (Aβ) burdening. Multidisciplinary studies, spanning from the tissue to the molecular level, are needed to assess how neuron/astrocyte interactions are influenced by aging. Our study addressed this requirement by joining fluorescence-lifetime imaging microscopy/phasor multiphoton analysis with confocal microscopy, implemented with a novel method to separate spectrally overlapped immunofluorescence and Aβ autofluorescence. By comparing data from young control rats, chronically inflamed rats, and old rats, we identified age-specific alterations of neuron/astrocyte interactions in the hippocampus. We found a correlation between Aβ aggregation (+300 and +800% of aggregated Aβ peptide in chronically inflamed and oldvs.control rats, respectively) and fragmentation (clasmatodendrosis) of astrocyte projections (APJs) (+250 and +1300% of APJ fragments in chronically inflamed and oldvs.control rats, respectively). Clasmatodendrosis, in aged rats, associates with impairment of astrocyte-mediated Aβ clearance (-45% of Aβ deposits on APJs, and +33% of Aβ deposits on neurons in oldvs.chronically inflamed rats). Furthermore, APJ fragments colocalize with Aβ deposits and are involved in novel Aβ-mediated adhesions between neurons. These data define the effects of Aβ deposition on astrocyte/neuron interactions as a key topic in AD biology.-Mercatelli, R., Lana, D., Bucciantini, M., Giovannini, M. G., Cerbai, F., Quercioli, F., Zecchi-Orlandini, S., Delfino, G., Wenk, G. L., Nos, D. Clasmatodendrosis and β-amyloidosis in aging hippocampus.

Ion channels regulate cell proliferation, differentiation, and migration in normal and neoplastic cells through cell-cell and cell-extracellular matrix (ECM) transmembrane receptors called integrins. K+ flux through the human ether-a-go-go-related gene 1 (hERG1) channel shapes action potential firing in excitable cells such as cardiomyocytes. Its abundance is often aberrantly high in tumors, where it modulates integrin-mediated signaling. We found that hERG1 interacted with the beta(1) integrin subunit at the plasma membrane of human cancer cells. This interaction was not detected in cardiomyocytes because of the presence of the hERG1 auxiliary subunit KCNE1 (potassium voltage-gated channel subfamily E regulatory subunit 1), which blocked the beta(1) integrin-hERG1 interaction. Although open hERG1 channels did not interact as strongly with beta(1) integrins as did closed channels, current flow through hERG1 channels was necessary to activate the integrin-dependent phosphorylation of Tyr(397) in focal adhesion kinase (FAK) in both normal and cancer cells. In immunodeficient mice, proliferation was inhibited in breast cancer cells expressing forms of hERG1 with impaired K+ flow, whereas metastasis of breast cancer cells was reduced when the hERG1/beta(1) integrin interaction was disrupted. We conclude that the interaction of beta(1) integrins with hERG1 channels in cancer cells stimulated distinct signaling pathways that depended on the conformational state of hERG1 and affected different aspects of tumor progression.

Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the single muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7+ satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration.

A combined transmission electron microscopy (TEM) and Fourier transform analysis has been performed on the secretory granules storing active peptides/proteins in serous cutaneous glands of n = 12 anuran species. Previous TEM investigation showed that the granules are provided with remarkable repeating substructures based on discrete subunits, arranged into a consistent framework. Furthermore, TEM findings revealed that this recurrent arrangement is acquired during a prolonged post-Golgian (or maturational) processing that affects the secretory product. Maturation leads to a variety of patterns depending on the degree of subunit clustering. This variety of recurrent patterns has been plotted into a range of frequency spectra. Through this quantitative approach, we found that the varying granule substructure can be reduced to a single mechanism of peptide/protein aggregation.

The possibility to induce myocardial regeneration by the activation of resident cardiac stem cells (CSCs) has raised great interest. However, to propose endogenous CSCs as therapeutic options, a better understanding of the complex mechanisms controlling heart morphogenesis is needed, including the cellular and molecular interactions that cardiomyocyte precursors establish with cells of the stromal compartment. In the present study, we co-cultured immature cardiomyocytes from neonatal mouse hearts with mouse bone marrow-derived mesenchymal stromal cells (MSCs) to investigate whether these cells could influence cardiomyocyte growth in vitro. We found that cardiomyocyte proliferation was enhanced by direct co-culture with MSCs compared with the single cultures. We also showed that the proliferative response of the neonatal cardiomyocytes involved the activation of Notch-1 receptor by its ligand Jagged-1 expressed by the adjacent MSCs. In fact, the cardiomyocytes in contact with MSCs revealed a stronger immunoreactivity for the activated Notch-intracellular domain (Notch-ICD) as compared with those cultured alone and this response was significantly attenuated when MSCs were silenced for Jagged-1. The presence of various cardiotropic cytokines and growth factors in the conditioned medium of MSCs underscored the contribution of paracrine mechanisms to Notch-1 up-regulation by the cardiomyocytes. In conclusions these findings unveil a previously unrecognized function of MSCs in regulating cardiomyocyte proliferation through Notch-1/Jagged-1 1 pathway and suggest that stromal-myocardial cell juxtacrine and paracrine interactions may contribute to the development of new and more efficient cell-based myocardial repair strategies. (C) 2011 Elsevier Ltd.

An understanding of the composition of natural and synthetic dyes used in ancient textiles and rugs is important for conservation purposes and to determine the origin of a textile or rug. Colorants or dyes are traditionally analysed by means of separation methods and ultraviolet–visible spectroscopic techniques that require sampling. This contribution focuses on establishing a fast, non-invasive, and in situ measurement procedure based on ultraviolet–visible–near infrared reflectance spectroscopic techniques for the characterization of dyes in ancient textiles or rugs manufactured in the Mediterranean basin and Middle East. For this purpose a collection of specimens on wool supports was prepared following ancient dyeing recipes. The samples were subsequently measured in the 230–2500 and 350–1700 nm ranges to create a spectroscopic database that was then compared with spectra acquired from textiles and rugs in order to characterize the dyes present.

Physiology and pathology have a big deal on tissue morphology, and the intrinsic spatial resolution of an atomic force microscope (AFM) is able to observe ultrastructural details. In order to investigate cellular and subcellular structures in histological sections with the AFM, we used a new simple method for sample preparation, i.e. chemical etching of semithin sections from epoxy resin-embedded specimens: such treatment appears to melt the upper layers of the embedding resin; thus, removing the superficial roughness caused by the edge of the microtome knife and bringing into high relief the biological structures hidden in the bulk. Consecutive ultrathin sections embedded in epoxy resin were observed with a transmission electron microscope (TEM) to compare the different imaging properties on the same specimen sample. In this paper we report, as an example, our AFM and TEM images of two different tissue specimens, rat pancreas and skeletal muscle fibres, showing that most of the inner details are visible with the AFM. These results suggest that chemical etching of histological sections may be a simple, fast and cost-effective method for AFM imaging with ultrastructural resolution.

Pulse temporal characterization is a fundamental task when operating a Ti:Sapphire ultrafast laser system for multiphoton microscopy applications. In the present report, an ultracompact autocorrelator setup and a simple procedure is reported to perform pulse width measurements at the focal plane of the microscope objective without the need of any further instrumentation, aside from a few optical elements, since the confocal microscope, detection, data acquisition, processing, and displaying capabilities are used. Microsc. Res. Tech. 63:27–33, 2004. © 2003 Wiley-Liss, Inc.

We have previously shown that sphingosine 1-phosphate (S1P) can induce intracellular Ca(2+) mobilization and cell contraction in C2C12 myoblasts and that the two phenomena are temporally unrelated. Although Ca(2+)-independent mechanisms of cell contraction have been the focus of numerous studies on Ca(2+) sensitization of smooth muscle, comparatively less studies have focused on the role that these mechanisms play in the regulation of skeletal muscle contractility. Phosphorylation and activation of myosin by Rho-dependent kinase mediate most of Ca(2+)-independent contractile responses. In the present study, we examined the potential role of Rho/Rho-kinase cascade activation in S1P-induced C2C12 cell contraction. First, we showed that depletion of Ca(2+), by pre-treatment with BAPTA, did not affect S1P-induced myoblastic contractility, whereas it abolished S1P-induced Ca(2+) transients. These results correlated with the absence of troponin C and with the immature cytoskeletal organization of these cells. Experimental evidence demonstrating the involvement of Rho pathway in S1P-stimulated myoblast contraction included: the activation/translocation of RhoA to the membrane in response to agonist-stimulation in cells depleted of Ca(2+) and the inhibition of dynamic changes of the actin cytoskeleton in cells where Rho functions had been inhibited either by overexpression of RhoGDI, a physiological inhibitor of GDP dissociation from Rho proteins, or by pretreatment with Y-27632, a specific Rho kinase inhibitor. Contribution of protein kinase C in this cytoskeletal rearrangement was also evaluated. However, the pretreatment with Gö6976 or rottlerin, specific inhibitors of PKC alpha and PKC delta, respectively, failed to inhibit the agonist-induced myoblastic contraction. Single particle tracking of G-actin fluorescent probe was performed to statistically evaluate actin cytoskeletal dynamics in response to S1P. Stimulation with S1P was also able to increase the phosphorylation level of myosin light chain II. In conclusion, our results strongly suggest that Ca(2+)-independent/Rho-Rho kinase-dependent pathways may exert an important role in S1P-induced myoblastic cell contraction.

In many cell systems, sphingosine 1-phosphate (SPP) increases cytosolic Ca2+concentration ([Ca2+]i) by acting as intracellular mediator and extracellular ligand. We recently demonstrated (Meacci E, Cencetti F, Formigli L, Squecco R, Donati C, Tiribilli B, Quercioli F, Zecchi-Orlandini S, Francini F, and Bruni P. Biochem J 362: 349–357, 2002) involvement of endothelial differentiation gene (Edg) receptors (Rs) specific for SPP in agonist-mediated Ca2+ response of a mouse skeletal myoblastic (C2C12) cell line. Here, we investigated the Ca2+ sources of SPP-mediated Ca2+ transients in C2C12 cells and the possible correlation of ion response to cytoskeletal rearrangement. Confocal fluorescence imaging of C2C12 cells preloaded with Ca2+ dye fluo 3 revealed that SPP elicited a transient Ca2+ increase propagating as a wave throughout the cell. This response required extracellular and intracellular Ca2+ pool mobilization. Indeed, it was significantly reduced by removal of external Ca2+, pretreatment with nifedipine (blocker of L-type plasma membrane Ca2+channels), and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-mediated Ca2+pathway inhibitors. Involvement of EdgRs was tested with suramin (specific inhibitor of Edg-3). Fluorescence associated with Ins(1,4,5)P3Rs and L-type Ca2+channels was evident in C2C12 cells. SPP also induced C2C12 cell contraction. This event, however, was unrelated to [Ca2+]i increase, because the two phenomena were temporally shifted. We propose that SPP may promote C2C12 cell contraction through Ca2+-independent mechanisms.

Sphingosine 1-phosphate (SPP) is a bioactive lipid that exerts multiple biological effects in a large variety of cell types, acting as either an intracellular messenger or an extracellular ligand coupled to Edg-family receptors (where Edg stands for endothelial differentiation gene). Here we report that in C2C12 myoblasts SPP elicited significant Ca2+ mobilization. Analysis of the process using a confocal laser-scanning microscope showed that the Ca2+ response occurred in a high percentage of cells, despite variations in amplitude and kinetics. Quantitative analysis of SPP-induced Ca2+ transients performed with a spectrophotofluorimeter showed that the rise in Ca2+ was strictly dependent on availability of extracellular Ca2+. Cell treatment with pertussis toxin partially prevented the Ca2+ response induced by SPP, indicating that Gi-coupled-receptors were involved. Indeed, SPP action was shown to be mediated by agonist-specific Edg receptors. In particular, suramin, an antagonist of the SPP-specific receptor Edg3, as well as down-regulation of Edg3 by cell transfection with antisense oligodeoxyribonucleotides (ODN), significantly reduced agonist-mediated Ca2+ mobilization. Moreover, an antisense ODN designed to inhibit Edg5 expression also decreased the SPP-induced rise in Ca2+, although to a lesser extent than that observed by inhibiting Edg3. On the contrary, the SPP response was unaffected in myoblasts loaded with antisense ODN specific for Edg1. Remarkably, the concomitant inhibition of Edg3 and Edg5 receptors abolished the SPP-induced Ca2+ increase, supporting the notion that Ca2+ mobilization in C2C12 cells induced by SPP is a receptor-mediated process that involves Edg3 and Edg5, but not Edg1.

Blocking visibility of a laser beam after a pair of safety goggles have been worn is always an unpleasant experience. Working blindly is hard, sometimes even dangerous, and safety could be again at risk. A safe, clear view of the laser beam path would be highly desirable. This paper presents a technique for laboratory laser safety, using a smartphone's camera and display, in conjunction with an augmented reality headset to allow clear viewing of laser experiments without any risk of laser eye injury. Use of the technique is demonstrated, and strengths and weaknesses of the solution are discussed.

Cell-to-cell contacts are crucial for cell differentiation. The promyogenic cell surface protein, Cdo, functions as a component of multiprotein clusters to mediate cell adhesion signaling. Connexin 43, the main connexin forming gap junctions, also plays a key role in myogenesis. At least part of its effects is independent of the intercellular channel function, but the mechanisms underlying are unknown. Here, using multiple optical approaches, we provided the first evidence that Cx43 physically interacts with Cdo to form dynamic complexes during myoblast differentiation, offering clues for considering this interaction a structural basis of the channel-independent function of Cx43. (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

We report on a new source able to provide probe pulses in the UV visible range and on the demonstration of its application to hyperspectral (fluorescence lifetime) imaging measurements. The source is able to generate UV (down to 300 nm) and blue light exploiting high-order mode propagation in a microstructured fiber pumped by a Ti:Sapphire laser. We believe that further optimization of pump wavelength, fiber length and fiber zero-dispersion wavelength could generate light well below 300 nm using a simple and stable set-up and become a useful tool for biomedical imaging. We demonstrated its versatility using the source for FLIM-FRET measurement a 460 nm and hyperspectral FRET-FLIM measurements. © 2014 SPIE.

An hybrid atomic force-scanning near-field optical microscope (AFM-SNOM) has been realized starting from a home-built AFM. The instrument uses a tetrahedral SiN tip for force and near field detection and is designed to provide, besides simple imaging, a full three-dimensional (3-D) mapping of force, friction and light intensity on the sample. High-resolution optical images of dielectric samples are presented together with curves describing the behavior of the collected optical intensity as a function of tip-object distance. AFM images and force-distance curves are reported as well and are compared with those obtained from the optical channel. Subwavelength features of the samples can be easily appreciated in both kinds of images, and the refractive index of the object can be computed from the attenuation constant of the tunneling light detected by the tip, with the AFM curve providing a convenient way of detecting the contact point. The spatial resolution of this kind of measurement is far better than that obtainable with any other index-measuring device. Homogeneous glass samples as well as monomode channel waveguides were analyzed in our experiments. The refractive index of several different prisms, made either of BK7 (n=1.519 at /spl lambda/=532 nm) or of SF58 (n=1.932) glass, could be measured with our near-field technique. As for the channel waveguides, the accuracy of our measurements lets us detect their presence and lateral extension in the substrate but is not yet sufficient to allow the reconstruction of their index profile.

We report on the systematic characterization of photoluminescence (PL) lifetimes in NV- and NV0 centers in 2 MeV H+ implanted type Ib diamond samples by means of a time correlated single photon counting (TCSPC) microscopy technique. A dipole-dipole resonant energy transfer model was applied to interpret the experimental results, allowing a quantitative correlation of the concentration of both native (single substitutional nitrogen atoms) and ion-induced (isolated vacancies) PL-quenching defects with the measured PL lifetimes. The TCSPC measurements were carried out in both frontal (i.e. laser beam probing the main sample surface along the same normal direction of the previously implanted ions) and lateral (i.e. laser beam probing the lateral sample surface orthogonally with respect to the same ion implantation direction) geometries. In particular, the latter geometry allowed a direct probing of the centers lifetime along the strongly nonuniform damage profiles of MeV ions in the crystal. The extrapolation of empirical quasi-exponential decay parameters allowed the systematic estimation of the mean quantum efficiency of the centers as a function of intrinsic and ion-induced defect concentration, which is of direct relevance for the current studies on the use of diamond color centers for photonic applications., 39 pages, 12 figures

A polarization interferometer which measures optical path differences with improved accuracy control is presented. The approach makes use of four signals in quadrature, computing the phase with an algorithm insensitive to laser power drifts. Experimental results of the interferometer’s performance are given under laser warmup conditions, and with long‐term monitoring of optical path differences.

In this paper we report imaged neuronal rat cells in a confocal laser scanning microscope by simultaneous generation of the three requested wavelengths obtained by a UV-extended supercontinuum source. This is to the best of our knowledge that such a measure was performed using a microstructured fiber pumped by a standard Ti:Sapphire laser. We observed efficient UV light generation when a novel pumping scheme was used. The pump wavelength is close to the zero-dispersion wavelength of the fiber first high-order mode and offset axial pumping is used. By tuning the pump wavelength and power level we were able to generate mW-power levels in the visible wavelength interval down and of about hundreds of microwatt in the UV wavelength interval down to 300 nm. The pump alignment was very simple and very stable. We believe that further optimization of pump wavelength, fiber length and fiber zero-dispersion wavelength could generate light well below 300 nm using a simple and stable set-up. To demonstrate the potentiality of this technique we imaged neuronal rat cells in a confocal laser scanning microscope by simultaneous generation of the three requested wavelengths.

The purpose of this study was to investigate the efficacy of Integra (R), an artificial dermal matrix used as a dermal template for skin regeneration, to form a multifunctional scaffold with human bone marrow-derived mesenchymal stem cells (hMSCs) and platelet-rich plasma (PRP) for tissue engineering and regenerative technology. First, we showed that PRP, used as a supplement for growth medium, represented an optimal substitute for animal serum as well as a source of multiple growth factors, was able to satisfactorily support cell viability and cell proliferation and influence stemness gene expression in hMSCs. Moreover, Integra appeared to be a suitable substrate for hMSCs colonization, as judged by two-photon microscopy combined with fluorescence lifetime imaging (FLIM) and confocal analysis. The cells were then seeded on Integra + PRP for 24 and 48 h. Notably, in these conditions, the seeded cells exhibited a greater aptitude to colonize the scaffold, showed improved cell adhesion and spreading as compared with those cultured on Integra alone, and acquired a fibroblast-like phenotype, indicating that the bioengineered scaffold provided an appropriate environment for cellular growth and differentiation. In conclusion, these results, although preliminary, provide clues for the design of new therapeutic strategies for skin regeneration, consisting in the combination of mesenchymal stem cells with engineered biomaterials. Copyright (C) 2011 John Wiley & Sons, Ltd.

The interaction of amyloid aggregates with the cell plasma membrane is currently considered among the basic mechanisms of neuronal dysfunction in amyloid neurodegeneration. We used amyloid oligomers and fibrils grown from the yeast prion Sup35p, responsible for the specific prion trait [PSI(+)], to investigate how membrane lipids modulate fibril interaction with the membranes of cultured H-END cells and cytotoxicity. Sup35p shares no homology with endogenous mammalian polypeptide chains. Thus, the generic toxicity of amyloids and the molecular events underlying cell degeneration can be investigated without interference with analogous polypeptides encoded by the cell genome. Sup35 fibrils bound to the cell membrane without increasing its permeability to Ca(2+). Fibril binding resulted in structural reorganization and aggregation of membrane rafts, with GM1 clustering and alteration of its mobility. Sup35 fibril binding was affected by GM1 or its sialic acid moiety, but not by cholesterol membrane content, with complete inhibition after treatment with fumonisin B1 or neuraminidase. Finally, cell impairment resulted from caspase-8 activation after Fas receptor translocation on fibril binding to the plasma membrane. Our observations suggest that amyloid fibrils induce abnormal accumulation and overstabilization of raft domains in the cell membrane and provide a reasonable, although not unique, mechanistic and molecular explanation for fibril toxicity.

Mesenchymal stromal cells (MSCs) are the leading cell candidates in the field of regenerative medicine. These cells have also been successfully used to improve skeletal muscle repair/regeneration; however, the mechanisms responsible for their beneficial effects remain to be clarified. On this basis, in the present study, we evaluated in a co-culture system, the ability of bone-marrow MSCs to influence C2C12 myoblast behavior and analyzed the cross-talk between the two cell types at the cellular and molecular level. We found that myoblast proliferation was greatly enhanced in the co-culture as judged by time lapse videomicroscopy, cyclin A expression and EdU incorporation. Moreover, myoblasts immunomagnetically separated from MSCs after co-culture expressed higher mRNA and protein levels of Notch-1, a key determinant of myoblast activation and proliferation, as compared with the single culture. Notch-1 intracellular domain and nuclear localization of Hes-1, a Notch-1 target gene, were also increased in the co-culture. Interestingly, the myoblastic response was mainly dependent on the paracrine release of vascular endothelial growth factor (VEGF) by MSCs. Indeed, the addition of MSC-derived conditioned medium (CM) to C2C12 cells yielded similar results as those observed in the co-culture and increased the phosphorylation and expression levels of VEGFR. The treatment with the selective pharmacological VEGFR inhibitor, KRN633, resulted in a marked attenuation of the receptor activation and concomitantly inhibited the effects of MSC-CM on C2C12 cell growth and Notch-1 signaling. In conclusion, this study provides novel evidence for a role of MSCs in stimulating myoblast cell proliferation and suggests that the functional interaction between the two cell types may be exploited for the development of new and more efficient cell-based skeletal muscle repair strategies.

RIASSUNTO L'articolo presenta i risultati dello studio compiuto su un cannocchiale sul quale si trova la firma di Galileo.

Optical sensors have a large impact in the fields of life science research, drug discovery and medical diagnostics. The recent advances in nanotechnology and photonics have led to a new generation of nanotools, capable of probing even the single cell: it has already been demonstrated that nanobiosensors can detect biochemical targets and proteins inside living single cells. Here we provide a brief overview of the field of nanoprobes consisting of tapered, metal-coated optical fibers having nanosize tips, such as those which were originally developed for use in near-field optical microscopy. Moreover we present some preliminary results concerning the characterization of the experimental sensing system which exploits such nanoprobes for intracellular biomedical diagnostics. The feasibility of using the Fluorescence Lifetime Imaging Microscopy (FLIM) technique as a dynamic diagnostics tool with these nanoprobes has been demonstrated.

SUMMARY The results of a series of optical tests recently performed on the Galileo's telescopes are reported. In particular, evidence is given of the interferometric quality of the lenses, nearly attaining optical perfection.

Some sources of error with multiphase homodyne interferometry are reviewed. A major advantage over the classic two-channel approach is that the inaccuracies that originate from laser-power fluctuations and drifts are shown to be automatically compensated for by proper adjustment of the light beams.

This chapter covers, in a very brief and introductory way, some of the fundamental aspects of modern optical microscopy, such as contrast techniques, spatial resolution topics, and high-speed acquisition solutions. It provides a sort of guided tour to introduce the crowded and complex world of optical microscopy, whose topics are treated in detail in the other chapters of this book.

We report on the application of an optically tunable coherent white light source, based on the supercontinuum generation from microstructured optical fibres, to fluorescence lifetime imaging (FLIM) microscopy and Foerster resonance energy transfer (FRET). A prototype lens based on monotonic longitudinal chromatic aberration is used for tuning the supercontinuum wavelengths from 400 to 1000 nm and acts as an axial monochromator, suitable for fibre delivery in confocal microscopy.

The statistics of partially developed speckle patterns produced by reflection under coherent illumination can be used to determine the scattering properties of a mirror. The theory of the approach is presented, showing that in proper conditions the standard deviation of the speckle-field distribution expresses the finish grade of the reflecting surface. Interferometric measurements are described, and experiments on selected samples are reported.

Monolayers and bilayers of lipid mixtures self-assembled on mercury form spontaneously gel-phase (solid ordered, s(o)) and liquid-ordered (l(o)) microdomains, thanks to the fluidity imparted to these films by the liquid metal support. The differential capacity of the hydrocarbon tail region of monolayers of mixtures of two lipid components of high and low transition temperature T-m, increases during the transition from the liquid disordered (l(d)) phase to the coexistence of l(d) and s(o) phases. Addition of cholesterol to this binary mixture causes a decrease in differential capacity. This behavior is explained by regarding the capacity as a measure of the total perimeter of the s(o) microdomains, due to the mismatch between these microdomains and the l(d) phase. Cholesterol removes this mismatch by converting the anisotropic s(o) microdomains into isotropic l(o) microdomains (rafts). This allows differential capacity measurements by electrochemical impedance spectroscopy to follow phase transitions in lipid mixtures. The coexistance of l(d), l(o) and s(o) phases is confirmed by images of a distal lipid monolayer self-assembled on top of a thiolipid monolayer tethered to a mercury microcap, by using two-photon fluorescence lifetime imaging microscopy (2P-FLIM).

We report on the application of a simple white light source based on the supercontinuum generation from commercial photonic crystal fibres to confocal fluorescence microscopy and fluorescence lifetime imaging (FLIM) microscopy. The coherent white light can be tuned by varying the wavelength and intensity of the pump, a Ti:Sapphire laser. There are several advantages jn the use of SC sources: spatially coherent white radiation, tuning ranges of approximately 400 nm, high brightness, a robust compact system (potentially all-fibre) and relatively low cost. Being pulsed, SC sources are suitable for FLIM and may be used for multiple excitation.

In this paper we report the results of measurements performed by FLIM on the photoreceptor of Euglenagracilis. This organelle consists of optically bistable proteins, characterized by two thermally stable isomeric forms: A(498,) non fluorescent and B(462), fluorescent. Our data indicate that the primary photoevent of Euglena photoreception upon photon absorption consists of two contemporaneous different phenomena: an intramolecular photo-switch (i.e., A(498) becomes B(462)), and a intermolecular and unidirectional Forster-type energy transfer. During the FRET process, the fluorescent B(462) form acts as donor for the non-fluorescent A(498) form of the protein nearby, which acts as acceptor. We hypothesize that in nature these phenomena follow each other with a domino progression along the orderly organized and closely packed proteins in the photoreceptor layer(s), modulating the isomeric composition of the photoreceptive protein pool. This mechanism guarantees that few photons are sufficient to produce a signal detectable by the cell.

The use of compact disc (CD) pickups in optical profilometry is a well-established practice. The instruments currently available on the market are, however, expensive both to purchase and to maintain. This expense is mainly due to the high cost of the scanning system, and it makes the use of low-cost pickups fruitless. Moreover, translation stages are bulky, slow, and in most applications neither necessary nor desirable. We present a one-dimensional profilometer, which uses a CD pickup as both the sensor and the actuator. Beam scanning of the sample is in fact performed by the objective lens tracking motor. The device is cheap, fast, compact, light, and a valuable solution for fluid and hard-to-access surface profiling.

Androgens and the androgen receptor (AR) are involved both in early tumorigenesis of prostate cancer (PCa) and in androgen-refractory disease. The role of AR signalling has also been highlighted by the fusion gene TMPRSS2:ERG recently identified in the majority of PCa. Several data indicate that re-expression of AR in PCa cell lines confers a less aggressive phenotype. We observed that re-expression of AR in the AR-negative cells PC3 decreases anchorage-independent growth and Matrigel invasiveness of PC3-AR cells where plasma membrane interaction between AR and EGFR led to an interference with downstream signalling and internalization of activated EGFR. Our data evidenced a shift of EGFR internalization pathway from the clathrin-coated pit one mediating signalling and recycling of EGFR to the lipid raft-mediated one mainly involved in lysosomal degradation of EGFR. These effects involved an altered recruitment to EGFR of the adaptor proteins Grb2 and c-Cbl followed by a reduced ubiquitination of EGFR. Our preliminary results suggest that in PC3-AR cells a pool of classical AR is located within cholesterol-rich membrane microdomains (namely as lipid rafts) and a population of EGFR is within cholesterol-rich membrane microdomains too. However, AR and EGFR membrane interaction that is increased by rapid androgen signalling is not within cholesterol-rich membrane microdomains. Our data enlighten that the crosstalk between genotropic and non-genotropic AR signalling interferes with signalling of EGFR in response to ligand leading to a lower invasive phenotype of AR-positive PCa cells.

A simple and fast interferometric measurement mainly for applications in thermal IR frequency range is presented. This method makes use of a single detector and a single spatial scan and thus reduces both measuring time and hardware complexity. The principle of operator is frequency coding of one transverse spatial dimension.

Phase-shifting interferometry techniques are used to directly investigate the first order statistics of real and imaginary parts of fully developed speckle fields. With reference to classic random walk models, the major assumptions and steps leading to the observed distributions are illustrated on experimental data sets.

Partially developed speckle fields produced by a weak scatterer are studied with digital phase-shifting interferometry techniques. Data acquisition and processing is described, and laboratory results are presented

Nonlinear optical microscopy is a relatively new and rapidly growing field of optical engineering, where Ti:sapphire ultrafast laser sources and technologies are finding a wide application. Diagnostic techniques addressed to this kind of application have been widely developed in the last few years. Research efforts have been focused on the evaluation and eventual correction of laser pulse duration widening due to group velocity dispersion of microscope optics, and devices have been specially designed to perform second-order autocorrelation measurements at the objective focal plane. In the present work, innovative, simple setups and procedures are reported that make the best use of all the facilities and characteristics of the microscope itself, so that only a few optical components are needed to temporal characterize the laser pulse at the specimen plane.

In our experiments, we have successfully managed to produce optical waveguides in polished LiF crystals by using He/sup +/ ion beam irradiation. The beam was masked by a metal window, producing large irradiated stripes about 1 mm wide. Irradiation energy was chosen to be 1.1 MeV, since it allowed to produce 3 /spl mu/m thick waveguides with a parabolic colour centre creation and thus refractive index profile, which was very suitable for characterisation purposes.

Sphingosine 1-phosphate (S1P) is a bioactive lipid that is abundantly present in the serum and mediates multiple biological responses. With the aim of extending our knowledge on the role played by S1P in the regulation of cytoskeletal reorganization, native as well as C2C12 myoblasts stably transfected with green fluorescent protein (GFP)-tagged α- and β-actin constructs were stimulated with S1P (1 μM) and observed under confocal and multiphoton microscopes. The addition of S1P induced the appearance of actin stress fibres and focal adhesions through Rho- and phospholipase D (PLD)-mediated pathways. The cytoskeletal response was dependent on the extracellular action of S1P through its specific surface receptors, since the intracellular delivery of the sphingolipid by microinjection was unable to modify the actin cytoskeletal assembly. Interestingly, it was revealed by whole-cell patch-clamp that S1P-induced stress fibre formation was associated with increased ion currents and conductance through stretch-activated channels (SACs), thereby suggesting a possible regulatory role for organized actin in channel sensitivity. Experiments aimed at stretching the plasma membrane of C2C12 cells, using the cantilever of an atomic force microscope, indicated that there was a Ca2+ influx through putative SACs. In conclusion, the present data suggest novel mechanisms of S1P signalling involving actin cytoskeletal reorganization and Ca2+ elevation through SACs that might influence myoblastic functions.

Multiphoton microscopy is a relatively new and rapidly growing field of applied optics where Ti:Sapphire ultrafast laser sources and related technology find a wide application. Laser beam diagnostic techniques specially devoted to this kind of application has been widely developed in these last years. Research efforts have been addressed to the evaluation and the eventual correction of the laser pulse duration widening due to group velocity dispersion caused by the microscope optics. Temporal characterization is thus a fundamental task when operating a ultrafast laser system for multiphoton microscopy applications and it is carried out by means of autocorrelators specially designed to perform pulse width measurements at the focus of the microscope objective. In the present communication, an innovative autocorrelator set-up and a simple metrological procedure are reported.

Sphingosine 1-phosphate (S1P) activates a subset of plasma membrane receptors of the endothelial differentiation gene family (EdgRs) in many cell types. In C2C12 myoblasts, exogenous S1P elicits Ca2+ transients by activating voltage-independent plasma membrane Ca2+ channels and intracellular Ca2+-release channels. In this study, we investigated the effects of exogenous S1P on voltage-dependent L-type Ca2+ channels in skeletal muscle fibers from adult mice. To this end, intramembrane charge movements (ICM) and L-type Ca2+ current (I(Ca)) were measured in single cut fibers using the double Vaseline-gap technique. Our data showed that submicromolar concentrations of S1P (100 nM) caused a approximately 10-mV negative shift of the voltage threshold and transition voltages of q(gamma) and q(h) components of ICM, and of I(Ca) activation and inactivation. Biochemical studies showed that EdgRs are expressed in skeletal muscles. The involvement of EdgRs in the above S1P effects was tested with suramin, a specific inhibitor of Edg-3Rs. Suramin (200 microM) significantly reduced, by approximately 90%, the effects of S1P on ICM and I(Ca), suggesting that most of S1P action occurred via Edg-3Rs. Moreover, SIP at concentration above 10 microM elicited intracellular Ca2+ transients in muscle fibers loaded with the fluorescent Ca2+ dye Fluo-3, as detected by confocal laser scanning microscopy.