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Ensemble and single particle studies of the excitation power density (P)-dependent upconversion luminescence (UCL) of core and core-shell β-NaYF4:Yb,Er upconversion nanoparticles (UCNPs) doped with 20% Yb3+ and 1% or 3% Er3+ performed over a P regime of 6 orders of magnitude reveal an increasing contribution of the emission from high energy Er3+ levels at P > 1 kW/cm2. This changes the overall emission color from initially green over yellow to white. While initially the green and with increasing P the red emission dominate in ensemble measurements at P < 1 kW/cm2, the increasing population of higher Er3+ energy levels by multiphotonic processes at higher P in single particle studies results in a multitude of emission bands in the ultraviolet/visible/near infrared (UV/vis/NIR) accompanied by a decreased contribution of the red luminescence. Based upon a thorough analysis of the P-dependence of UCL, the emission bands activated at high P were grouped and assigned to 2–3, 3–4, and 4 photonic processes involving energy transfer (ET), excited-state absorption (ESA), cross-relaxation (CR), back energy transfer (BET), and non-radiative relaxation processes (nRP). This underlines the P-tunability of UCNP brightness and color and highlights the potential of P-dependent measurements for mechanistic studies required to manifest the population pathways of the different Er3+ levels.

International audience; Two homometallic ytterbium dimers are prepared and their solution-state photoluminescence and upconversion properties are investigated. Both complexes exhibit two-photon cooperative luminescence upconversion in the visible region (lambda(em) approximate to 510 nm) upon excitation into the near-infrared Yb F-2(5/2)

The podand-type ligand L, based on a tertiary amine substituted by three pyridyl-6-phosphonic acid functions, forms hydrated complexes with Ln3+ cations. The luminescence properties of the YbL complex were studied in D2O as a function of the pD and temperature. In basic conditions, increases in the luminescence quantum yield and the excited state lifetime of the Yb centered emission associated with the 2F5/2 → 2F7/2 transition were observed and attributed to a change in the hydration number from two water molecules in the first coordination sphere of Yb at acidic pH to a single one in basic conditions. Upon the addition of TbCl3 salts to a solution containing the YbL complex in D2O, heteropolynuclear Yb/Tb species formed, and excitation of the Yb at 980 nm resulted in the observation of the typical visible emission of Tb as a result of a cooperative upconversion (UC) photosensitization process. The UC was further evidenced by the quadratic dependence of the UC emission as a function of the laser power density.

International audience; Upconversion nanoparticles are a promising class of materials for bioimaging. Their attractive optical properties are influenced by their crystalline phase, size, and morphology resulting from the preparation method employed. Herein, we synthesized NaYF4:Yb,Er nanocrystals by the co-precipitation method followed by a heat treatment step. The influence of the treatment temperature and the type of reactor on the phase transition from the α-cubic to the β-hexagonal phase was investigated. We found that before the phase transition, the size and shape of α-NaYF4:Yb,Er nanocrystals evolved due to a coalescence mechanism with microtubular reactors. In contrast to the flask system, we also observed an anisotropic growth of β-NaYF4:Yb,Er nanorods in microtubes whose length can be controlled by adjusting the temperature. The diameter of microtubes plays a critical role in the shape evolution of the final products from spheres to nanorods. The type of reactor and thus the resulting temperature gradient could be considered as a promising parameter to control the phase, shape and size of UCNPs without changing the chemical composition.

International audience; Hyperbranched poly(N-(2-hydroxypropyl) methacrylamide)s grafted on upconversion nanoparticles (UCNPs) were prepared by surface-initiated self-condensing vinyl RAFT copolymerization from the surface of UCNPs modified with a RAFT chain transfer agent. The effect of the grafting density in RAFT chain transfer agent on UCNPs and the concentration in transmer were investigated. The propagation of the polymerization was strongly affected by termination and transfer reactions associated to the high local concentration in propagating chains due to the immobilization of the polymer chains at the surface of UCNPs and increased number of active centers during the polymerization, but also to the hopping and rolling mechanisms related to the surface-initiated RAFT polymerization. The development of such nanohybrids opens the way to novel nano-objects for biomedical imaging and theranostics.

Piling up excited states to reach upconversion (UC) is severely restricted by vibrational quenching mechanisms, especially when one looks at discrete molecular entities in solution. By carefully controlling the supramolecular assembly processes resulting from the strong electrostatic interactions between negatively charged Yb complexes and Tb3+ cations in aqueous solutions, we engineered the formation of heteropolynuclear complexes of [(YbL)2Tb x] compositions ( x = 1 and 2). These edifices display a phenomenon of cooperative photosensitization UC with green emission of the Tb cations upon NIR excitation at 980 nm in the Yb absorption band. The photophysical properties of the complexes were carefully investigated by steady-state and time-resolved luminescence experiments in D2O, allowing one to quantify the impact of the composition and pD of the solution on the emission intensity as well as clarifying the exact cooperative photosensitization upconversion mechanism. Using optimized conditions, the energy transfer UC process could be observed for the first time in nondeuterated water with discrete molecular compounds.

Single-particle luminescence microscopy is a powerful method to extract information on biological systems that is not accessible by ensemble-level methods. Upconversion nanoparticles (UCNPs) are a particularly promising luminophore for single-particle microscopy as they provide stable, non-blinking luminescence and allow the avoidance of biological autofluorescence by their anti-Stokes emission. Recently, ensemble measurements of diluted aqueous dispersions of UCNPs have shown the instability of luminescence over time due to particle dissolution-related effects. This can be especially detrimental for single-particle experiments. However, this effect has never been estimated at the individual particle level. Here, the luminescence response of individual UCNPs under aqueous conditions is investigated by quantitative wide-field microscopy. The particles exhibit a rapid luminescence loss, accompanied by large changes in spectral response, leading to a considerable heterogeneity in their luminescence and band intensity ratio. Moreover, the dissolution-caused intensity loss is not correlated with the initial particle intensity or band ratio, which makes it virtually unpredictable. These effects and the subsequent development of their heterogeneity can be largely slowed down by adding millimolar concentrations of sodium fluoride in buffer. As a consequence, the presented data indicate that microscopy experiments employing UCNPs in an aqueous environment should be performed under conditions that carefully prevent these effects.

Retinoic acid receptors (RARs) as a functional heterodimer with retinoid X receptors (RXRs), bind a diverse series of RA-response elements (RAREs) in regulated genes. Among them, the non-canonical DR0 elements are bound by RXR–RAR with comparable affinities to DR5 elements but DR0 elements do not act transcriptionally as independent RAREs. In this work, we present structural insights for the recognition of DR5 and DR0 elements by RXR–RAR heterodimer using x-ray crystallography, small angle x-ray scattering, and hydrogen/deuterium exchange coupled to mass spectrometry. We solved the crystal structures of RXR–RAR DNA-binding domain in complex with the Rarb2 DR5 and RXR–RXR DNA-binding domain in complex with Hoxb13 DR0. While cooperative binding was observed on DR5, the two molecules bound non-cooperatively on DR0 on opposite sides of the DNA. In addition, our data unveil the structural organization and dynamics of the multi-domain RXR–RAR DNA complexes providing evidence for DNA-dependent allosteric communication between domains. Differential binding modes between DR0 and DR5 were observed leading to differences in conformation and structural dynamics of the multi-domain RXR–RAR DNA complexes. These results reveal that the topological organization of the RAR binding element confer regulatory information by modulating the overall topology and structural dynamics of the RXR–RAR heterodimers.

Reverse transcriptase (RT) of human immunodeficiency virus-1 (HIV-1) is a multifunctional enzyme that catalyzes the conversion of the single stranded viral RNA genome into double-stranded DNA, competent for host-cell integration. RT is endowed with RNA- and DNA-dependent DNA polymerase activity and DNA-directed RNA hydrolysis (RNase H activity). As a key enzyme of reverse transcription, RT is a key target of currently used highly active antiretroviral therapy (HAART), though RT inhibitors offer generally a poor resistance profile, urging new RT inhibitors to be developed. Using single molecule fluorescence approaches, it has been recently shown that RT binding orientation and dynamics on its substrate play a critical role in its activity. Currently, most in vitro RT activity assays, inherently end-point measurements, are based on the detection of reaction products by using radio-labeled or chemically modified nucleotides. Here, we propose a simple and continuous real-time Förster resonance energy transfer (FRET) based-assay for the direct measurement of RT's binding orientation and polymerase activity, with the use of conventional steady-state fluorescence spectroscopy. Under our working conditions, the change in binding orientation and the primer elongation step can be visualized separately on the basis of their opposite fluorescence changes and their different kinetics. The assay presented can easily discriminate non-nucleoside RT inhibitors from nucleoside RT inhibitors and determine reliably their potency. This one-step and one-pot assay constitutes an improved alternative to the currently used screening assays to disclose new anti-RT drugs and identify at the same time the class to which they belong.

Upconverting nanoparticles (UCNPs) are luminophores that have been investigated for a multitude of biological applications, notably low-background imaging, high-sensitivity assays, and cancer theranostics. In these applications, they are frequently used as a donor in resonance energy transfer (RET) pairs. However, because of the peculiarity and non-linearity of their luminescence mechanism, their behavior as a RET pair component has been difficult to predict quantitatively, preventing their optimization for subsequent applications. In this article, we assembled UCNP–organic dye RET systems and investigated their luminescence decays and spectra, with varying UCNP sizes and quantities of dyes grafted onto their surface. We observed an increase in RET efficiency with lower particle sizes and higher dye decoration. We also observed several unexpected effects, notably a quenching of UCNP luminescence bands that are not resonant with the absorption of organic dyes. We proposed a semi-empirical Monte Carlo model for predicting the behavior of UCNP-organic dye systems, and validated it by comparison with our experimental data. These findings will be useful for the development of more accurate UCNP-based assays, sensors, and imaging agents, as well as for optimization of UCNP–organic dye RET systems employed in cancer treatment and theranostics.

Optical transmission through arrays of subwavelength apertures is studied systematically in different metals over the optical spectral range in comparable conditions. The results provide an absolute comparison and thereby highlight the influence of the dielectric constants, surface excitations, interband transitions and different loss mechanisms. Only noble metals such as Ag, Au and Cu give rise to significant transmission in the optical regime. Hole arrays in W transmit very poorly, as expected, since W cannot sustain surface plasmons in the optical range.

International audience; Being a scanning microscopy, Stimulated Emission Depletion (STED) needs to be parallelized for fast wide-field imaging. Here, we achieve large parallelization of STED microscopy using well-designed Optical Lattice (OL) for depletion, together with a fast camera for detection. Depletion optical lattices with 100 intensity "zeros" are generated by four-beam interference. Scanning only a unit cell, as small as 290 nm by 290 nm, of the depletion OL is sufficient for STED imaging. The OL-STED microscopy acquires super-resolution images with 70 nm resolution and at a rate of 12.5 frames per image.

Recent developments in stimulated emission depletion (STED) microscopy achieved nanometer scale resolution and showed great potential in live cell imaging. Yet, STED nanoscopy techniques are based on single point-­‐scanning. This constitutes a drawback for wide field imaging, since the gain in spatial resolution requires dense pixelation and hence long recording times. Here we achieve massive parallelization of STED nanoscopy using wide-­‐field excitation together with well-­‐designed optical lattices for depletion and a fast camera for detection. Acquisition of large field of view super-­‐resolved images requires scanning over a single unit cell of the optical lattice which can be as small as 290 nm*290nm. Interference STED (In-­‐STED) images of 2.9 μm* 2.9 μm with resolution down to 70 nm are obtained at 12.5 frames per second. The development of this technique opens many prospects for fast wide-­‐field nanoscopy.

During reverse transcription, the HIV-1 RNA is converted by the reverse transcriptase (RT) into proviral DNA. RT is assisted by the HIV-1 nucleocapsid (NCp7) protein that notably increases the ability of RT to synthesize DNA through pause sites. Using single molecule FRET, we monitored the NCp7 effect on the binding of RT to nucleic acid sequences corresponding to two different pause sites. NCp7 was found to modify the distribution of RT orientations on the oligonucleotides and decrease the residence time of RT on one of the pause sites. These results give direct insight into the NCp7 molecular mechanism in reverse transcription.

We analyze the progressive introduction of disorder in periodic subwavelength hole arrays. Two models of disorder are discussed from their associated Fourier transforms and correlation functions. The optical transmission properties of the corresponding arrays are closely related with the evolutions of structure factors, as experimentally detailed. Remarkably, the optical properties of random arrays are not in general equal to those of the single hole as a result of short-range correlations corresponding to hole-to-hole interactions. These correlations are due to packing constraints that are controlled through the careful generation of random patterns. For high density pattern, short-range order can take over long-range order associated with the periodic array.

We investigate transmission efficiency and finite size effects for the subwavelength hole arrays. Experiments and simulations show how the finite size effects depend strongly on the hole diameter. The transmission efficiency reaches an asymptotic upper value when the array is larger than the surface plasmon propagation length on the corrugated surface. By comparing the transmission of arrays with that of the corresponding single holes, the relative enhancement is found to increase as the hole diameter decreases. In the conditions of the experiments the enhancement is one to two orders of magnitude but there is no fundamental upper limit to this value.

During HIV-1 reverse transcription, the single-stranded RNA genome is converted into proviral double stranded DNA by Reverse Transcriptase (RT) within a reverse transcription complex composed of the genomic RNA and a number of HIV-1 encoded proteins, including the nucleocapsid protein NCp7. Here, we developed a one-step and one-pot RT polymerization assay. In this in vitro assay, RT polymerization is monitored in real-time by Förster resonance energy transfer (FRET) using a commercially available doubly-labeled primer/template DNA. The assay can monitor and quantify RT polymerization activity as well as its promotion by NCp7. Z-factor values as high as 0.89 were obtained, indicating that the assay is suitable for high-throughput drug screening. Using Nevirapine and AZT as prototypical RT inhibitors, reliable IC50 values were obtained from the changes in the RT polymerization kinetics. Interestingly, the assay can also detect NCp7 inhibitors, making it suitable for high-throughput screening of drugs targeting RT, NCp7 or simultaneously, both proteins.

4 pages, 4 figures.-- et al., The extraordinary optical transmission through periodic arrays of subwavelength holes has been studied extensively since it was first reported in 1998, owing to both its fundamental implications and its technological potential. The picture of the underlying mechanism that emerges from most of the theoretical studies is a resonant process assisted by surface electromagnetic modes, such as surface plasmons. However, these studies consider an infinite array of holes. By combining experiment and theory, we have analysed the influence of the inherent finite size of the arrays and report here the unexpected spatial distribution of light as it emerges from the arrays. This distribution is strongly anisotropic and extremely sensitive to the angle of incidence of the impinging light. The behaviour can be explained by a model that takes into account the asymmetry induced by the array edges, and the effects this has on the emission pattern across the array., Financial support by the EC under Project Nos. FP6-NMP4-CT-2003-505699 (Surface Plasmon Photonics) and FP6-2002-IST-1-507879 (Plasmo-Nano-Devices) is gratefully acknowledged.

Arrays of subwavelength holes in metal film show enhanced transmission when compared to what is expected from classical diffraction. It appears that this phenomenon is crucially related to surface plasmon polaritons. By their nature, surface plasmon polaritons are particularly sensitive to the optical properties of the metal film. In this work we relate in the optical regime these properties on the enhanced transmission.

The opening of the light extraction cone of a high index substrate (GaP) is demonstrated using plasmonic gratings. We show that the excitation of surface plasmons on the metal grating leads to the extraction of light otherwise undergoing total internal reflection in the substrate with high efficiency. This effect has an immediate application in the context of light emitting diodes, where resonant metal gratings are promising for the design of dual purpose electric contact/extraction structures.