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Microcirculation plays a key role in the maintenance and hemodynamics of tissues and organs also due to its extensive interaction with the immune system. A critical limitation of state-of-the-art clinical techniques to characterize the blood flow is their lack of the spatial resolution required to scale down to individual capillaries. On the other hand the study of the blood flow through auto- or cross-correlation methods fail to correlate the flow speed values with the morphological details required to describe an intricate network of capillaries. Here we propose to use a newly developed technique (FLICS, FLow Image Correlation Spectroscopy) that, by employing a single raster-scanned xy-image acquired in vivo by confocal or multi-photon excitation fluorescence microscopy, allows the quantitative measurement of the blood flow velocity in the whole vessel pattern within the field of view, while simultaneously maintaining the morphological information on the immobile structures of the explored circulatory system. Fluorescent flowing objects produce diagonal lines in the raster-scanned image superimposed to static morphological details. The flow velocity is obtained by computing the Cross Correlation Function (CCF) of the intensity fluctuations detected in pairs of columns of the image. The whole analytical dependence of the CCFs on the flow speed amplitude and the flow direction has been reported recently. We report here the derivation of approximated analytical relations that allows to use the CCF peak lag time and the corresponding CCF value, to directly estimate the flow speed amplitude and the flow direction. The validation has been performed on Zebrafish embryos for which the flow direction was changed systematically by rotating the embryos on the microscope stage. The results indicate that also from the CCF peak lag time it is possible to recover the flow speed amplitude within 13% of uncertainty (overestimation) in a wide range of angles between the flow and th

This Brief focuses on the synthesis, functionalization techniques, optical properties and biomedical application of gold nanostars (GNS). Various facilities of gold nanostars synthesis as well as functionalization of GNS with PEG, organic dyes, bioactive compounds are discussed. The authors discuss physical origin of the Localized Surface Plasmon Resonances and the way the nano-environment affects them. The implication of the LSPR of gold nanostars surface enhanced Raman scattering is also discussed. The emphasis has been done on the application of GNS for current and emerge needs of medicine, biology and pharmacy. Moreover, properties of gold nanostars as contrast agents for in vivo imaging and interaction of GNS with cells are also discussed in this Brief.

We have previously addressed experimentally blood fluidodynamics in microcapillaries by coupling optical microscopy to pixelated detection. By computing the Cross-Correlation Function (CCF) of signals coming from pixels at a distance along the flow we obtained information on the flow speed and direction. The extension of these experiments to more complex systems with high branching of capillaries and/or inverted flows needs a theoretical investigation that we present here. We focus first on straight capillaries and harmonic flows between a minimum Vmin ≠0 and a maximum Vmax flow speed. The CCF shows multiple peaks at lag times that correspond closely to the maximum and minimum flow speeds. The general analytical expression of the CCF is given, the position of its maxima are discussed by means of geometrical considerations and numerical analysis and an experimental validation are presented. The second case that we study is the flow in the branches of a y-shaped junction in a microcapillary. By simply modeling the branching in laminar flow (low Reynold numbers) and assuming a smooth transition of speeds along the branches we derive a simple numerical model to compute the trajectories of micro-beads. We estimate the flow speed in the branches by computing the CCFs between linear regions of interest set perpendicular to the axes of the branches.

Monolayers of photothermally responsive gold nanostars on PEI-coated surfaces display two localized surface plasmon resonances (LSPRs) in the near-IR region that can be laser-irradiated either separately, obtaining two different T jumps, or simultaneously, obtaining a T jump equal to the sum of those obtained with separate irradiations.

The optical monitoring of multiple single neuron activities requires high-throughput parallel acquisition of signals at millisecond temporal resolution. To this aim, holographic two-photon microscopy (2PM) based on spatial light modulators (SLMs) has been developed in combination with standard laser scanning microscopes. This requires complex coordinate transformations for the generation of holographic patterns illuminating the points of interest. We present a simpler and fully digital setup (SLM-2PM) which collects three-dimensional two-photon images by only exploiting the SLM. This configuration leads to an accurate placement of laser beamlets over small focal volumes, eliminating mechanically moving parts and making the system stable over long acquisition times. Fluorescence signals are diffraction limited and are acquired through a pixelated detector, setting the actual limit to the acquisition rate. High-resolution structural images were acquired by raster-scanning the sample with a regular grid of excitation focal volumes. These images allowed the selection of the structures to be further investigated through an interactive operator-guided selection process. Functional signals were collected by illuminating all the preselected points with a single hologram. This process is exemplified for high-speed (up to 1 kHz) two-photon calcium imaging on acute cerebellar slices.

Anisotropic metallic nanoparticles have been devised as powerful potential tools for in vivo imaging, photothermal therapy, and drug delivery thanks to plasmon-enhanced absorption and scattering cross sections, ease in synthesis and functionalization, and controlled cytotoxicity. The rational design of all these applications requires the characterization of the nanoparticles intracellular trafficking pathways. In this work, we exploit live-cell time-lapse confocal reflectance microscopy and image correlation in both direct and reciprocal space to investigate the intracellular transport of branched gold nanostars (GNSs). Different transport mechanisms, spanning from pure Brownian diffusion to (sub-)ballistic superdiffusion, are revealed by temporal and spatio-temporal image correlation spectroscopy on the tens-of-seconds timescale. According to these findings, combined with numerical simulations and with a Bayesian (hidden Markov model-based) analysis of single particle tracking data, we ascribe the superdiffusive, subballistic behavior characterizing the GNSs dynamics to a two-state switching between Brownian diffusion in the cytoplasm and molecular motor-mediated active transport. For the investigation of intermittent-type transport phenomena, we derive an analytical theoretical framework for Fourier-space image correlation spectroscopy (kICS). At first, we evaluate the influence of all the dynamic and kinetic parameters (the diffusion coefficient, the drift velocity, and the transition rates between the diffusive and the active transport regimes) on simulated kICS correlation functions. Then we outline a protocol for data analysis and employ it to derive whole-cell maps for each parameter underlying the GNSs intracellular dynamics. Capable of identifying even simpler transport phenomena, whether purely diffusive or ballistic, our intermittent kICS approach allows an exhaustive investigation of the dynamics of GNSs and biological macromolecules.

The twelve-membered tetraazamacrocyclic ligand L1 bears an appended lipoic acid unit, whose disulphide ring is an efficient grafting moiety for the surface of gold nanostars (GNS). The GNS that were used featured a localized surface plasmon resonance (LSPR) absorption at ∼800 nm, i.e. in the near infrared (NIR). We investigated different approaches for coating them with the Cu2+ complex of L1. While the direct reaction of [CuL1]2+ with as-prepared GNS led to aggregation, an initial coating step with polyethyleneglycol-thiol (PEG-SH) was found to be advantageous. Displacement reactions were carried out on pegylated GNS either with [CuL1]2+, directly generating [Cun(L1@GNS)]2n+, or with void L1, thus obtaining L1@GNS that coordinates Cu2+ in a second step. In both cases, even with a large excess of the competing disulphide moiety, only partial displacement of PEG-SH is observed, obtaining ca. 500-1500 [CuL1]2+ per GNS depending on the conditions, with PEG-SH remaining in the [Cun(L1@GNS)]2n+ hybrids and imparting them with remarkable stability. Comparison of the photothermal and two-photon luminescence (TPL) properties of the GNS between the pegylated GNS and [Cun(L1@GNS)]2n+ revealed that the grafted copper complex does not change them to any extent. Finally, the stability against demetallation and transmetallation of the complexes, as well as the fast kinetics of complexation of the monodispersed macrocycle and of L1@GNS, have been examined, suggesting [Cun(L1@GNS)]2n+ as a device capable of TPL optical tracking and NIR photothermal therapy and as a possible agent for PET imaging.

The increased resistances to conventional antibiotics determine a strong need for new antibacterials, and specific syntheses at the nanoscale promise to be helpful in this field. A novel Zinc-doped Copper oxide nanocomposite (nZn-CuO) has been recently sonochemically synthesized and successfully tested also against multi-drug resistant bacteria. After synthesis and characterization of the physicochemical properties, the new nZn-CuO is here evaluated by the Frog Embyo Teratogenesis Assay-Xenopus test for its toxicological potential and this compared with that of nCuO and nZnO synthesized under the same conditions. No lethal effects are observed, while malformations and growth retardation slightly increase after nZn-CuO exposure. Nevertheless, these effects are smaller than those of nZnO. NP uptake by embryo tissues increase significantly with increasing NP concentrations, while no significant accumulation and adverse effects are seen after exposure to soluble Cu2+ and Zn2+ at the concentrations dissolved from the NPs. Key oxidative response genes are upregulated by nZn-CuO, as well as by nCuO and nZnO, suggesting the common mechanism of action. Considering the enhanced biocidal activity shown by the nanocomposite, together with the results presented in this study, we can affirm that the doping of the metal oxide nanoparticles should be considered a useful tool to engineer a safer nano-antibacterial.

This paper describes the scientific aims and potentials as well as the preliminary technical design of RUDE, an innovative tool for multi-disciplinary investigations in a wide field of scientific, technological and industrial applications. IRIDE will be a high intensity "particles factory", based on a combination of high duty cycle radio-frequency superconducting electron linacs and of high energy lasers. Conceived to provide unique research possibilities for particle physics, for condensed matter physics, chemistry and material science, for structural biology and industrial applications, IRIDE will open completely new research possibilities and advance our knowledge in many branches of science and technology. [RIDE is also supposed to be realized in subsequent stages of development depending on the assigned priorities.

In practice, the catchment hydrologist is often confronted with the task of predicting discharge without having the needed records for calibration. Here, we report the discharge predictions of 10 modellers - using the model of their choice - for the man-made Chicken Creek catchment (6 ha, northeast Germany, Gerwin et al., 2009b) and we analyse how well they improved their prediction in three steps based on adding information prior to each following step. The modellers predicted the catchment's hydrological response in its initial phase without having access to the observed records. They used conceptually different physically based models and their modelling experience differed largely. Hence, they encountered two problems: (i) to simulate discharge for an ungauged catchment and (ii) using models that were developed for catchments, which are not in a state of landscape transformation. The prediction exercise was organized in three steps: (1) for the first prediction the modellers received a basic data set describing the catchment to a degree somewhat more complete than usually available for a priori predictions of ungauged catchments; they did not obtain information on stream flow, soil moisture, nor groundwater response and had therefore to guess the initial conditions; (2) before the second prediction they inspected the catchment on-site and discussed their first prediction attempt; (3) for their third prediction they were offered additional data by charging them pro forma with the costs for obtaining this additional information. Hollander et al. (2009) discussed the range of predictions obtained in step (1). Here, we detail the modeller's assumptions and decisions in accounting for the various processes. We document the prediction progress as well as the learning process resulting from the availability of added information. For the second and third steps, the progress in prediction quality is evaluated in relation to individual modelling experience and costs of ad, QC 20140811

We describe a novel method (FLICS, FLow Image Correlation Spectroscopy) to extract flow speeds in complex vessel networks from a single raster-scanned optical xy-image, acquired in vivo by confocal or two-photon excitation microscopy. Fluorescent flowing objects produce diagonal lines in the raster-scanned image superimposed to static morphological details. The flow velocity is obtained by computing the Cross Correlation Function (CCF) of the intensity fluctuations detected in pairs of columns of the image. The analytical expression of the CCF has been derived by applying scanning fluorescence correlation concepts to drifting optically resolved objects and the theoretical framework has been validated in systems of increasing complexity. The power of the technique is revealed by its application to the intricate murine hepatic microcirculatory system where blood flow speed has been mapped simultaneously in several capillaries from a single xy-image and followed in time at high spatial and temporal resolution.

Biomedical issues in vasculogenesis and cardiogenesis require methods to follow hemodynamics with high spatial (micrometers) and time (milliseconds) resolution. At the same time, we need to follow relevant morphogenetic processes on large fields of view. Fluorescence cross-correlation spectroscopy coupled to scanning or wide-field microscopy meets these needs but has limited flexibility in the excitation pattern. To overcome this limitation, we develop here a two-photon two-spots setup coupled to an all-reflective near-infrared (NIR) optimized scanning system and to an electron multiplying charge-coupled device. Two NIR laser spots are spaced at adjustable micron-size distances (1 to 50 ìm) by means of a Twyman-Green interferometer and repeatedly scanned on the sample, allowing acquisition of information on flows at 4 ms-3 ìm time-space resolution in parallel on an extended field of view. We analyze the effect of nonhomogeneous and variable flow on the cross-correlation function by numerical simulations and show exemplary application of this setup in studies of blood flow in zebrafish embryos in vivo. By coupling the interferometer with the scanning mirrors and by computing the cross-correlation function of fluorescent red blood cells, we are able to map speed patterns in embryos' vessels. © 2014 Society of Photo-Optical Instrumentation Engineers.

Monolayers of gold nanostars (GNS) are grafted on mercaptopropyltrimethoxysilane-coated glass slides. In the formed monolayers the localized surface plasmon resonance of GNS can be tuned in the 700–1100 nm range. Upon laser excitation of the nearIR LSPR an efficient photothermal response is observed, inducing local hyperthermia and efficient killing of Staphylococcus aureus biofilms. © 2014 The Royal Society of Chemistry.

In order to investigate the spatiotemporal organization of neuronal activity in local microcircuits, techniques allowing the simultaneous recording from multiple single neurons are required. To this end, we implemented an advanced spatial-light modulator two-photon microscope (SLM-2PM). A critical issue for cerebellar theory is the organization of granular layer activity in the cerebellum, which has been predicted by single-cell recordings and computational models. With SLM-2PM, calcium signals could be recorded from different network elements in acute cerebellar slices including granule cells (GrCs), Purkinje cells (PCs) and molecular layer interneurons. By combining WCRs with SLM-2PM, the spike/calcium relationship in GrCs and PCs could be extrapolated toward the detection of single spikes. The SLM-2PM technique made it possible to monitor activity of over tens to hundreds neurons simultaneously. GrC activity depended on the number of spikes in the input mossy fiber bursts. PC and molecular layer interneuron activity paralleled that in the underlying GrC population revealing the spread of activity through the cerebellar cortical network. Moreover, circuit activity was increased by the GABA-A receptor blocker, gabazine, and reduced by the AMPA and NMDA receptor blockers, NBQX and APV. The SLM-2PM analysis of spatiotemporal patterns lent experimental support to the time-window and center-surround organizing principles of the granular layer

The interaction among leukocytes is at the basis of the innate and adaptive immune-response and it is largely ascribed to direct cell-cell contacts. However, the exchange of a number of chemical stimuli (chemokines) allows also non-contact interaction during the immunological response. We want here to evaluate the extent of the effect of the non-contact interactions on the observed leukocyte-leukocyte kinematics and their interaction duration. To this aim we adopt a simplified mean field description inspired by the Keller-Segel chemotaxis model, of which we report an analytical solution suited for slowly varying sources of chemokines. Since our focus is on the non-contact interactions, leukocyte-leukocyte contact interactions are simulated only by means of a space dependent friction coefficient of the cells. The analytical solution of the Keller-Segel model is then taken as the basis of numerical simulations of interactions between leukocytes and their duration. The mean field interaction force that we derive has a time-space separable form and depends on the chemotaxis sensitivity parameter as well as on the chemokines diffusion coefficient and their degradation rate. All these parameters affect the distribution of the interaction durations. We draw a successful qualitative comparison between simulated data and sets of experimental data for DC-NK cells interaction duration and other kinematic parameters. Remarkably, the predicted percentage of the leukocyte-leukocyte interactions falls in the experimental range and depends (≅25% increase) upon the chemotactic parameter indicating a non-negligible direct effect of the non-contact interaction on the leukocyte interactions. © 2013 Gritti et al.

Fluorophores useful for STimulated Emission Depletion (STED) spectroscopy must fulfill strict requirements on depletion efficiency and photostability. These parameters determine the effective resolution of STED imaging. Resolution is typically measured on 30-80 nm spheres heavily decorated with STED bright fluorophores, limiting the possibility to estimate the true resolution achievable on a specific dye. Here we show how single molecule STED microscopy provides an estimate of the fluorophore stimulated emission cross section and of its photostability under STED irradiation. Fluorescein, a green and a yellow mutant of GFP, are tested, and the results are discussed and compared to those obtained with Chromeo488-covered 80 nm spheres on a commercial continuous-wave STED microscope.

Five-branched gold nanostars are obtained using Triton X-100 in a seed-growth synthesis. They have the uncommon feature of two intense localized surface plasmon resonances (LSPRs) in the 600–900 and 1100–1600 nm ranges. Both LSPRs convert laser radiation into heat, offering two photothermally active channels in the NIR and SWIR ranges. © 2013 The Royal Society of Chemistry.

Magnetic nanoparticles have emerged as important players in current research in modern medicine since they can be used in medicine for diagnosis and/or therapeutic treatment of diseases. Among many therapeutic applications of iron-based nanoparticles, drug delivery and photothermal therapy are of particular interest. At cellular level their uptake has been studied and the mechanism by which nanoparticles enter into the cell has important implication not only for their fate but also for their impact on the biological systems. We present here a dynamic investigation of interaction of biocompatible iron oxide nanoparticles coated with L-3,4-dihydroxyphenylalanine and labeled with tetra-methylrhodamine-5/6- isothiocyanate with lung epithelial cells. Our data show that after macropinocytosis-mediated internalization, nanoparticles in form of vesicles approach the nucleus and converge in the more acidic compartments of the cells in a microtubuledependent manner. During progression the nanoparticles aggregate. Finally, we have demonstrated that a converging laser radiation on the cells, causes the increase in the local temperature and thus damages the cells, suggesting that these nanoparticles may be applied for photothermal therapy studies. Copyright © 2013 American Scientific Publishers All rights reserved.

We developed an all-optical method to measure the temperature on gold (nanorods and nanostars) and magnetite nanoparticles under near-infrared and radiofrequency excitation by monitoring the excited state lifetime of Rhodamine B that lies within ≅20 nm from the nanoparticle surface. We reached high temperature sensitivity (0.029 ± 0.001 ns/ C) and low uncertainty (±0.3 C). Gold nanostars are ≅3 and ≅100 times more efficient than gold nanorods and magnetite nanoparticles in inducing localized hyperthermia. © 2013 American Chemical Society.

The armory of GFP mutants available to biochemists and molecular biologists is huge. Design and selection of mutants are usually driven by tailored spectroscopic properties, but some key aspects of stability, folding and dynamics of selected GFP variants still need to be elucidated. We have prepared, expressed and characterized three H148 mutants of the highly fluorescent variant GFPmut2. H148 is known to be involved in the H-bonding network surrounding the chromophore, and all the three mutants, H148G, H148R and H148K, show increased pKa values of the chromophore. Only H148G GFPmut2 (Mut2G) gave good expression and purification yields, indicating that position 148 is critical for efficient folding in vivo. The chemical denaturation of Mut2G was monitored by fluorescence emission, absorbance and far-UV circular dichroism spectroscopy. The mutation has little effect on the spectroscopic properties of the protein and on its stability in solution. However, the unfolding kinetics of the protein encapsulated in wet nanoporous silica gels, a system that allows to stabilize conformations that are poorly or only transiently populated in solution, indicate that the unfolding pathway of Mut2G is markedly different from the parent molecule. In particular, encapsulation allowed to identify an unfolding intermediate that retains a native-like secondary structure despite a destructured chromophore environment. Thus, H148 is a critical residue not only for the chromophoric and photodynamic properties, but also for the correct folding of GFP, and its substitution has great impact on expression yields and stability of the mature protein