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X-ray fluorescence (XRF) and reflectance spectroscopy (RS) are commonly used for the characterization of painting materials. It is well known that the former provides the chemical fingerprint of the pictorial layers, while the latter returns the molecular description of the pigments constituting the uppermost layers. Even if these two techniques cannot unveil the stratigraphy, their synergetic application well describes the materials employed for realizing the panels and represents a key turn for non-invasive scientific analysis of works of art. However, the potential of the cross-comparison between XRF and RS is not fully exploited yet. The measurement points often barely match, and they are usually few isolated spots spread over the whole surface of the painting; these facts limit the mutual exchange of information between the data sets and can lead to losing details. In this scenario, XRF mapping (MA-XRF) and hyperspectral reflectance imaging (HRI) provide a connection channel that promises to be a decisive tool to strengthen the relationship between X-ray fluorescence and reflectance spectroscopy and, therefore, to deepen the knowledge about the case studies. Due to the spatial localization of the information they contain, the maps provide not only a straightforward reference for comparing the data but also a three-dimensional collection of elemental and molecular images. By applying computer vision and statistical methods such as spectral angle mapper (SAM), it is possible to implement an innovative approach that exploits the elemental features, obtained from XRF spectra, to improve the comprehension of the molecular aspects given by RS, and vice versa. Once we discussed the main issues behind our approach, we applied it to analyze the painting Chariot Race by Giorgio De Chirico (1928–1929, oil on canvas, Pinacoteca di Brera, Milan, Italy). The results reflect the complexity of the painting, and even if only some of the spectra identified by the method as peculiar are ascribable to recognizable pigments, the mutual correspondence between elemental distributions and SAM maps defines a mixture of materials that matches the description given by the artist in his “Small Treatise on Pictorial Technique” (De Chirico in Abscondita, 2019).

Uncovering the underdrawings (UDs), the preliminary sketch made by the painter on the grounded preparatory support, is a keystone for understanding the painting's history including the original project of the artist, the pentimenti (an underlying image in a painting providing evidence of revision by the artist) or the possible presence of co-workers’ contributions. The application of infrared reflectography (IRR) has made the dream of discovering the UDs come true: since its introduction, there has been a growing interest in the technology, which therefore has evolved leading to advanced instruments. Most of the literature either report on the technological advances in IRR devices or present case studies, but a straightforward method to improve the visibility of the UDs has not been presented yet. Most of the data handling methods are devoted to a specific painting or they are not user-friendly enough to be applied by non-specialized users, hampering, thus, their widespread application in areas other than the scientific one, e.g., in the art history field. We developed a computer-assisted method, based on principal component analysis (PCA) and image processing, to enhance the visibility of UDs and to support the art-historians and curators’ work. Based on ImageJ/Fiji, one of the most widespread image analysis software, the algorithm is very easy to use and, in principle, can be applied to any multi- or hyper-spectral image data set. In the present paper, after describing the method, we accurately present the extraction of the UD for the panel “The Holy Family with St. Anne and the Young St. John” and for other four paintings by Luini and his workshop paying particular attention to the painting known as “The Child with the Lamb”.

The project “Leonardesque Artists beyond the Visible” was planned on the 500th anniversary of the death of Leonardo da Vinci, with the occasion of an exhibition held in Milan in 2019, where more than twenty works made by Leonardesque masters have been put on display. Among them, five representative paintings made by his closest pupils were selected for a comprehensive and multidisciplinary project. Portable non-invasive imaging and spectroscopy techniques were applied to supply useful information to scholars but also to the wider public: description of the material composition of the pigments, of the preparation and of the binders, existence or absence of underdrawing, and identification of the painters’ technique and style. Particular attention was paid also to the image processing techniques, mostly for hyperspectral and radiographic data, to get the most from both innovative and traditional techniques. Results highlighted for each author a peculiar painting technique showing hidden features such as pentimenti and the panel preparation methods, pigments, binders and varnishes.

Reflectance Spectroscopy (RS) and Fiber Optics Reflectance Spectroscopy (FORS) are well-established techniques for the investigation of works of art with particular attention to paintings. Most modern museums put at the disposal of their research groups portable equipment that, together with the intrinsic non-invasiveness of RS and FORS, makes possible the in situ collection of reflectance spectra from the surface of artefacts. The comparison, performed by experts in pigments and painting materials, of the experimental data with databases of reference spectra drives the characterization of the palettes and of the techniques used by the artists. However, this approach requires specific skills and it is time consuming especially if the number of the spectra to be investigated becomes large as is the case of Hyperspectral Reflectance Imaging (HRI) datasets. The HRI experimental setups are multi-dimensional cameras that associate the spectral information, given by the reflectance spectra, with the spatial localization of the spectra over the painted surface. The resulting datasets are 3D-cubes (called hypercubes or data-cubes) where the first two dimensions locate the spectrum over the painting and the third is the spectrum itself (i.e., the reflectance of that point of the painted surface versus the wavelength in the operative range of the detector). The capability of the detector to simultaneously collect a great number of spectra (typically much more than 10,000 for each hypercube) makes the HRI datasets large reservoirs of information and justifies the need for the development of robust and, possibly, automated protocols to analyze the data. After the description of the procedure designed for the data acquisition, we present an analysis method that systematically exploits the potential of the hypercubes. Based on Spectral Angle Mapper (SAM) and on the manipulation of the collected spectra, the algorithm handles and analyzes thousands of spectra while at the same time it supports the user to unveil the features of the samples under investigation. The power of the approach is illustrated by applying it to Quarto Stato, the iconic masterpiece by Giuseppe Pellizza da Volpedo, held in the Museo del Novecento in Milan (Italy).

In the context of paintings characterization, X-Ray Fluorescence (XRF) combined with Fibre Optics Reflectance Spectroscopy (FORS) is a well-known methodology. The latter allows to classify the pigments constituting the external layers (few microns) of the painting while the former provides the fingerprint of the main chemical elements present in almost all layers (tens of microns) of the sample. The synergetic application of XRF and FORS is a key turn for non-invasive scientific analysis of works of art since it exhaustively describes the pigments employed by the masters and supplies a first insight into their distribution on the panels. In this scenario, Optical Microscopy (OM) applied directly on the painting surface, without any type of sampling, is traditionally deemed as a descriptive tool for showing microscopic features such as craquelure, brushstrokes or painting material distribution. It can be eventually expendable as a qualitative help during the discussion of spectroscopic data, but not considered fundamental. Nonetheless, the application of image processing protocols can make OM a concrete support for the characterization of paintings. In this work a new image analysis strategy has been introduced, its potential has been demonstrated studying different flesh tones from the panel “The Holy Family with St. Anne and the young St. John” by Bernardino Luini (Pinacoteca Ambrosiana Collection of Milan) and, finally, it has been discussed how OM could quantitatively complete XRF and FORS analysis. The results show that optical microscopy, other than provide a glance of the investigated surface spots, effectively integrates the description given by the spectroscopic techniques and, properly managed, can be a useful resource for solving ambiguous cases.

The paper discusses data concerning the secondary manufacture technology of a set of opaque coloured early Islamic mosaic glass tesserae from the qasr of Khirbet al-Mafjar (Jericho, Palestine). Archaeological contextualisation of the site had allowed attributing these finds to the Umayyad occupational phase of the building, and an in-depth study of the composition of the glassy matrix had provided evidence of a double supply of glass from Egypt and the Syro-Palestinian coast occurring in the production of the base glass intended to be used for the manufacture of mosaic tesserae. Here, a multi-methodological approach has been carried out to characterise colourants and opacifiers: visible reflectance spectroscopy (VIS-RS), optical microscopy (OM), scanning electron microscopy coupled with energy dispersion analysis (SEM-EDS), micro-Raman spectroscopy (micro-Raman) and X-ray diffraction (XRD) were performed on the opaque tesserae. Moreover, either optically stimulated luminescence (OSL) or thermoluminescence (TL) protocols for luminescence dating were applied on selected samples, with the aim of relating luminescence properties with the geochemical features of the glass tesserae, in the perspective of deepening the studies towards the absolute dating, an unquestionable help to the stimulating challenge of investigating ancient glass manufacture. Tin-based, phosphorus-based and copper-based opacifiers were identified, and the achieved results suggest the use of the same opacifiers and colouring agents regardless of the different base glass. Furthermore, data obtained by TL and OSL protocols revealed useful and stimulating potentialities these techniques could have in dating opaque glasses.

This paper deals with a Thermoluminescence (TL) study of ancient mosaic tesserae from the qasr (winter residence) of Khirbat al-Mafjar, amazing palace of the Islamic caliphs located in the plain of Jericho. In literature, works dealing with the dating of mosaic tesserae usually rely on the dosimetric properties of the microcrystalline inclusions in the silica matrix rather than on the matrix itself. On the other hand, in the last decades, commercial glasses have been demonstrated to be suitable for retrospective accidental dosimetry. In this work, we applied a protocol widely used for the mobile phones’ glasses, the so-called “pre-bleached with blue LEDs” protocol, to evaluate the archeological dose absorbed by some Khirbat al-Mafjar tesserae in order to confirm their dating based on archaeological evidence. As for commercial glasses, the TL signal from ancient tesserae presents anomalous fading and it is light sensitive. The experimental protocol circumvents these problems isolating the thermally more stable TL signal with an optical pretreatment and allowing the determination of a fading curve for each analyzed sample. Using an integrative approach, we estimated the fading correction for these tesserae and we retrieved their ages. The results are partially in agreement with those hypothesized on historical ground and show good potentialities for the dating of amorphous archaeological glassy materials.

God the Father with Angels (about 1330, tempera on panel) by Giotto is the Gable of the altarpiece of Baroncelli Chapel in the church of Santa Croce in Florence. Very little is known about its history since the separation from the so-called Baroncelli Polyptych. Now at the San Diego Museum of Art, the Gable had never been studied by means of scientific methods before our team took the opportunity to during the exhibition “Giotto, l’Italia”, held in Milan. Exploiting the integration of different knowledge, technologies and resources of our team, we were able to provide data for understanding the organizational model of Giotto's workshop performing non-invasive analyses with portable instruments during closing hours of exhibition (four diagnostic campaigns, six hours of work/campaign, no interruption of the exhibition). The achieved results confirm the painting technique based on different layers of pigments, a technique already used by Giotto. Combining the effectiveness of scanning MA-XRF with the responsive of IR reflectography and IR false colour, we moved step by step toward the discovery of Giotto's palette for the flesh tones in God the Father with Angels. FORS and XRF single point analyses were performed on some selected areas too. The IR reflectography results support the hypothesis of a detailed underdrawing with both thin and flat brushstrokes. By applying image-processing algorithms to the collected reflectograms, we obtained quantitative objective measures supporting the hypothesis that a guide could have been used in the realization of human figures; this means the use of sketches for the face of “God the Father” and for the faces of angels.

The cell fate determinant Numb orchestrates tissue morphogenesis and patterning in developmental systems. In the human mammary gland, Numb is a tumor suppressor and regulates p53 levels. However, whether this function is linked to its role in fate determination remains unclear. Here, by exploiting an ex vivo system, we show that at mitosis of purified mammary stem cells (SCs), Numb ensures the asymmetric outcome of self-renewing divisions by partitioning into the progeny that retains the SC identity, where it sustains high p53 activity. Numb also controls progenitor maturation. At this level, Numb loss associates with the epithelial-to-mesenchymal transition and results in differentiation defects and reacquisition of stemness features. The mammary gland of Numb-knockout mice displays an expansion of the SC compartment, associated with morphological alterations and tumorigenicity in orthotopic transplants. This is because of low p53 levels and can be inhibited by restoration of Numb levels or p53 activity, which results in successful SC-targeted treatment.

The Giotto’s masterpiece God the Father with Angels, never investigated till now, was studied by our team of local researchers, involved in application of scientific methods for cultural heritage since many years. Exploiting the integration of different knowledges, technologies and resources of our team, we were able to provide data to understand the painting technique, the pigment used and the underdrawing of this Giotto’s painting. We performed the following non-invasive analyses: Macro-XRF scanning (MA-XRF), Fiber optic reflectance spectroscopy (FORS), high resolution IR scanning reflectography, infrared false color (IRFC). Only portable instrumentations were used, with operating times compatible with the opening hours of exhibition. In particular, the analytical campaign was the opportunity to test the portable IR scanning prototype based on a peculiar spherical scanning system characterized by light weight and low cost motorized head. The analytical results revealed a painting technique already used by Giotto and based on different superimposed pigment layers. By combining the effectiveness of scanning portable-XRF (pXRF) with the responsive of image spectroscopic analysis, we move step by step toward the discovery of Giotto’s palette, with particular attention to the flesh tones in God the Father with Angels. The imaging data support the hypothesis of a detailed underlying sketch that includes also a drawing characterized by larger brush signs; the use of patrones for the face of “God” was supposed thanks to comparison with other Giotto masterpieces.

The vascular system of Zebrafish embryos is studied by means of Fluorescence Correlation and Image Correlation Spectroscopy. The long term project addresses biologically relevant issues concerning vasculogenesis and cardiogenesis and in particular mechanical interaction between blood flow and endothelial cells. To this purpose we use Zebrafish as a model system since the transparency of its embryos facilitates morphological observation of internal organs in-vivo. The correlation analysis provides quantitative characterization of fluxes in blood vessels in vivo. We have pursued and compared two complementary routes. In a first one we developed a two-spots two-photon setup in which the spots are spaced at adjustable micron-size distances (1-40 μm) along a vessel and the endogenous (autofluorescence) or exogenous (dsRed transgenic erythrocytes) signal is captured with an EM-CCD and cross-correlated. In this way we are able to follow the morphology of the Zebrafish embryo, simultaneously measure the heart pulsation, the velocity of red cells and of small plasma proteins. These data are compared to those obtained by image correlations on Zebrafish vessels. The two methods allows to characterize the motion of plasma fluids and erythrocytes in healthy Zebrafish embryos to be compared in the future to pathogenic ones. © 2013 SPIE.

SummaryNatural killer (NK) cells have antitumor, antiviral, and antibacterial functions, and efforts are being made to manipulate them in immunotherapeutic approaches. However, their activation mechanisms remain poorly defined, particularly during bacterial infections. Here, we show that upon lipopolysaccharide or E. coli exposure, dendritic cells (DCs) produce three cytokines—interleukin 2 (IL-2), IL-18, and interferon β (IFN-β)—necessary and sufficient for NK cell activation. IFN-β enhances NK cell activation by inducing IL-15 and IL-15 receptor α not only in DCs but, surprisingly, also in NK cells. This process allows the transfer of IL-15 on NK cell surface and its cis presentation. cis-presented NK cell-derived and trans-presented DC-derived IL-15 contribute equally to optimal NK cell activation.

The lipopolysaccharide is the major component of Gram-negative bacteria outer membrane. LPS comprises three covalently linked regions: the lipid A, the rough core oligosaccharide, and the O-antigenic side chain determining serotype specificity. Wild-type LPS (sLPS) contains the O-antigenic side chain and is referred to as smooth. Rough LPS (rLPS) does not contain the O-side chain. Most wt bacteria and especially wt Enterobacteriaceae express prevalently the sLPS form although some truncated rLPS molecules always reach the external membrane. The two sLPS and rLPS forms are used almost indistinctly to study the effects on innate immune cells. Nevertheless, there is evidence that their mechanism of action may be different. For instance, while sLPS requires CD14 for the initiation of both MyD88-dependent and independent signal transduction pathways at least at low doses, rLPS leads to MyD88-dependent responses in the absence of CD14 even at low doses. Here we have identified additional differences in the signaling capacity of the two LPS species in the mouse. We have found that rLPS, diversely from sLPS, is capable of activating in dendritic cells (DCs) the Ca 2+/calcineurin and NFAT pathway in a CD14-independent manner, moreover it is also capable per se of activating the inflammasome and eliciting IL-1β secretion independent of the presence of additional stimuli required instead for sLPS. The ability of rLPS of activating the inflammasome in vitro has as a direct consequence a higher efficiency of rLPS-exposed DCs in activating natural killer (NK) cells compared to sLPS-exposed DCs. However, diversely from possible predictions, we found that the different efficiencies of the two LPS species in eliciting innate responses are almost nullified in vivo. Therefore, sLPS and rLPS induce nearly similar in vivo innate responses but with different mechanisms of signaling. © 2011 Elsevier B.V.

Under the action of near-infrared radiation, shape anisotropic gold nanoparticles emit two-photon luminescence and release heat. Accordingly, they have been proposed for imaging, photothermal therapies and thermo-controlled drug delivery. In all these applications particular care must be given to control the nanoparticle - cell interaction and the thermal efficiency of the nanoparticles, while minimizing their intrinsic cytotoxicity. We present here the characterization of the cell interaction of newly developed branched gold nanostars, obtained by laurylsulfobetaine-driven seed-growth synthesis. The study provides information on the size distribution, the shape anisotropy, the cellular uptake and cytotoxicity of the gold nanostars as well as their intracellular dynamic behavior by means of two-photon luminescence imaging, fluorescence correlation spectroscopy and particle tracking. The results show that the gold nanostars are internalized as well as the widely used gold nanorods and are less toxic under prolonged treatments. At the same time they display remarkable two-photon luminescence and large extinction under polarized light in the near-infrared region of the spectrum, 800-950 nm. Gold nanostars appear then a valuable alternative to other elongated or in-homogeneous nanoparticles for cell imaging. © 2012 American Chemical Society.

Fluorescence correlation spectroscopy (FCS) was first developed for biophysical studies in analogy with photon scattering correlation spectroscopy. Although it is mainly devoted to the study of freely diffusing particles, FCS is actually able to discern between different kinds of motions, such as diffusion, anomalous diffusion, or drift motions. The frontier application of FCS nowadays is in medical studies both within cells and on the cell membranes, and in the investigation of single molecules in solid matrices. In this field, FCS originated also image correlation spectroscopy methods. The whole field can be unified under the name of fluorescence fluctuation spectroscopy (FFS). We present here a short review of the theoretical bases of FFS under a unified vision and discuss some applications to the study of dynamics of nanoparticles in cells and to the investigation of the photodynamics of immobilized dyes.

The basic research in cell biology and in medical sciences makes large use of imaging tools mainly based on confocal fluorescence and, more recently, on non-linear excitation microscopy. Substantially the aim is the recognition of selected targets in the image and their tracking in time. We have developed a particle tracking algorithm optimized for low signal/noise images with a minimum set of requirements on the target size and with no a priori knowledge of the type of motion. The image segmentation, based on a combination of size sensitive filters, does not rely on edge detection and is tailored for targets acquired at low resolution as in most of the in-vivo studies. The particle tracking is performed by building, from a stack of Accumulative Difference Images, a single 2D image in which the motion of the whole set of the particles is coded in time by a color level. This algorithm, tested here on solid-lipid nanoparticles diffusing within cells and on lymphocytes diffusing in lymphonodes, appears to be particularly useful for the cellular and the in-vivo microscopy image processing in which few a priori assumption on the type, the extent and the variability of particle motions, can be done.

Dendritic cells (DCs) are key regulators of innate and adaptive immune responses that can be exploited in the immunological treatment of many type of cancers. Recently, we have demonstrated that lipopolysaccharide (LPS) is able to regulate DC life cycle through the activation of a CD14-dependet pathway [1]. Once activated with LPS, DCs become also able to prime Natural Killer (NK) cells to exert their anti-tumoral activity as demonstrated both in-vitro and in-vivo using mouse models in which melanoma tumors were sub-cute implanted. Using two-photon microscopy, we are currently extending these experiments to the direct investigation of the interaction of LPS-activated DCs with NK cells in in vivo condition at the level of peripheral lymph nodes.DCs and NK cells,labeled with different fluorescent markers, are tracked continuously in the lymph nodes while the structure of the lymph node is monitored by second harmonic generation microscopy. The analysis of the traces and the comparison of the experimental results to statistical and simulative models of the lymphocytes motion allows to elucidate their dynamic behavior at different times after the activation of the DCs shedding new light on the DCs - NK cells interaction.[1] Zanoni, I.; Ostuni, R.; Capuano, G.; Collini, M.; Caccia, M.; Ronchi, A.E.;Rocchetti, M.; Mingozzi, F.; Foti, M.; Chirico, G.; Costa, B.;Zaza, A.; Ricciardi-Castagnoli, P.; Granucci, F. (2009) CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation. Nature, 460:264-269.

Recent studies have demonstrated that dendritic cells (DCs) play a crucial role in the activation of Natural Killer cells (NKs) that are responsible for anti-tumor innate immune responses. The focus of this report is on the role of pathogen associated molecular pattern (PAMP) activated-DCs in inducing NK cell-mediated anti-tumor responses. Mice transplanted sub-cute (s.c.) with AK7 cells, a mesothelioma cell line sensitive to NK cell responses, are injected with fluorescent NK cells and DC activation is then induced by s.c. injection of Lipopolysaccharide (LPS). Using 4 dimensional tracking we follow the kinetic behavior of NK cells at the Draining Lymph-Node (DLN). As control, noninflammatory conditions are also evaluated. Our data suggest that NK cells are recruited to the DLN where they can interact with activated-DCs with a peculiar kinetic behavior: short lived interactions interleaved by rarer longer ones. We also found that the changes in the NK dynamic behavior in inflammatory conditions clearly affect relevant motility parameters such as the instantaneous and average velocity and the effective diffusion coefficient. This observation suggests that NK cells and activated-DCs might efficiently interact in the DLN, where cells could be activated. Therefore the interaction between activated-DCs and NK cells in DLN is not only a reality but it may be also crucial for the start of the immune response of the NKs. © 2010 Copyright SPIE - The International Society for Optical Engineering.

Toll-like receptors (TLRs) are the best characterized pattern recognition receptors. Individual TLRs recruit diverse combinations of adaptor proteins, triggering signal transduction pathways and leading to the activation of various transcription factors, including nuclear factor kappaB, activation protein 1 and interferon regulatory factors. Interleukin-2 is one of the molecules produced by mouse dendritic cells after stimulation by different pattern recognition receptor agonists. By analogy with the events after T-cell receptor engagement leading to interleukin-2 production, it is therefore plausible that the stimulation of TLRs on dendritic cells may lead to activation of the Ca(2+)/calcineurin and NFAT (nuclear factor of activated T cells) pathway. Here we show that mouse dendritic cell stimulation with lipopolysaccharide (LPS) induces Src-family kinase and phospholipase Cgamma2 activation, influx of extracellular Ca(2+) and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. We also show that LPS-induced NFAT activation via CD14 is necessary to cause the apoptotic death of terminally differentiated dendritic cells, an event that is essential for maintaining self-tolerance and preventing autoimmunity. Consequently, blocking this pathway in vivo causes prolonged dendritic cell survival and an increase in T-cell priming capability. Our findings reveal novel aspects of molecular signalling triggered by LPS in dendritic cells, and identify a new role for CD14: the regulation of the dendritic cell life cycle through NFAT activation. Given the involvement of CD14 in disease, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via CD14 is an important step towards the development of potential treatments involving interference with CD14 functions.

The interaction of the surface plasmons of gold nanoparticles (NP) a few nanometers in size with fluorophores can be used to engineer their fluorescence properties. This possibility can be exploited in principle to obtain nanodevices for protein-protein recognition. We studied different types of constructs based on gold NPs on which derivatives of fluorescein were bound. The interaction of this fluorophore with the gold surface plasmon resonances, mainly occurring through quenching, affects its excited-state lifetime that is measured by fluorescence burst analysis in standard solutions. The binding of proteins to the gold NPs through antigen-antibody recognition further modifies the dye excited-state lifetime. This change can therefore be used to measure the protein concentration. The data reported here indicate that one can measure the concentration of bovine serum albumine in solution with an apparent limit of detection of 5 +/- 2 pM.

Non-linear excitation microscopy is considered an ideal spectroscopic method for imaging thick tissues in vivo due to the reduced scattering of infrared radiation. Although imaging has been reported on brain neocortex at 600-800 μm of depth, much less uniform tissues, such as lymphonodes, are characterized by highly anisotropic light scattering that limits the penetration length. We show that the most severe limitation for deep imaging of lymphonodes appears to be the tissue scattering and the diffuse fluorescence emission of labeled cell (lymphocytes) in layers above the focusing plane. We report a study of the penetration depth of the infrared radiation in a model system and in ex vivo lymphonodes and discuss the possibility to apply Fourier filtering to the images in order to improve the observation depth. © 2008 EBSA.

The photon counting histogram (PCH) analysis of the fluorescence fluctuations provides the molecular brightness (ε) and the average number of fluorophores (〈Nn〉) in an open observation volume. PCH, which is based on the analysis of the whole of the photon counting histogram, has been recently improved by taking into account the detector dead time effect, which is relevant at high fluorescence rates. We investigate here the possibility of quantitatively applying the PCH analysis in the simplified form of photon moment analysis, in which only the first two moments of the photon counting histogram are computed. We have applied this analysis to low fluorescence signals from living cells in the presence of cell micro-movements and molecular photo-bleaching and describe a simple algorithm for its routine application. The algorithm has been tested on Saccharomyces Cerevisiæ (yeast) cells labeled with Dimethyl-pepep and Rhodamine 6G, and Chinese Hamster Ovary (CHO) cells stably expressing the regulatory subunit (RII) of protein kinase A fused to the cyan-emitting variant of GFP (CFP). Our statistical analysis allows us to estimate the local concentrations and the brightness of the fluorophores in different cellular compartments (nucleus, membrane, and cytoplasm) despite the occurrence of microscopic cell movements and significant photo-bleaching.

Green Fluorescent Protein (GFP) mutants are extensively used in optical microscopy studies of in vivo biological processes in cells. Nonetheless, blinking and bleaching of the GFP chromophore at the single molecule level greatly limits its usefulness. We have worked out what we think are the best experimental conditions for the use of the GFP mutant, GFP-mut2, as a single molecule marker in two-photon excitation measurements. We have measured molecular brightness, excited state lifetime, blinking and photo-bleaching times versus the two-photon excitation intensity on proteins embedded in silica gel matrices versus the excitation wavelength in the range 700-1,000 nm. Our results indicate that GFPmut2 can be employed as a long-lived reporter of biological processes. (C) 2005 Wiley-Liss, Inc.

Several lines of evidence indicate that differences in DNA repair capacity are an important source of variability in cancer risk. However, traditional assays for measurement of DNA repair activity in human samples are laborious and time-consuming. DNA glycosylases are the first step in base excision repair of a variety of modified DNA bases. Here, we describe the development of a new sensitive DNA glycosylase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. FCCS was applied to the measurement of uracil DNA glycosylase activity of human cell extracts and validated by comparison with standard gel electrophoresis assay. Our results indicate that FCCS can be adapted to efficient assays for DNA glycosylase activity in protein extracts from human cells. This method has a potential for the development of automated screening of large number of samples.