Targeted Single Particle Tracking with Upconverting Nanoparticles.

Single particle tracking (SPT) is a powerful technique for real-time microscopic visualization of the movement of individual biomolecules within or on the surface of living cells. However, SPT often suffers from the suboptimal performance of the photon-emitting labels used to tag the biomolecules of interest. For example, fluorescent dyes have poor photostability, while quantum dots suffer from blinking that hampers track acquisition and interpretation. Upconverting nanoparticles (UCNPs) have recently emerged as a promising anti-Stokes luminescent label for SPT. In this work, we demonstrated targeted SPT using UCNPs. For this, we synthesized 30 nm diameter doped UCNPs and coated them with amphiphilic polymers decorated with polyethylene glycol chains to make them water-dispersible and minimize their nonspecific interactions with cells. Coated UCNPs highly homogeneous in brightness (as confirmed by a single particle investigation) were functionalized by immunoglobulin E (IgE) using a biotin-streptavidin strategy. Using these IgE-UCNP SPT labels, we tracked high-affinity IgE receptors (FcεRI) on the membrane of living RBL-2H3 mast cells at 37 °C in the presence and absence of antigen and obtained good agreement with the literature. Moreover, we used the FcεRI-IgE receptor-antibody system to directly compare the performance of UCNP-based SPT labels to organic dyes (AlexaFluor647) and quantum dots (QD655). Due to their photostability as well as their backgroundless and continuous luminescence, SPT trajectories obtained with UCNP labels are no longer limited by the photophysics of the label but only by the dynamics of the system and, in particular, the movement of the label out of the field of view and/or focal plane.

Upconversion in a d-f [RuYb3] Supramolecular Assembly.

We have prepared a hetero-tetrametallic assembly consisting of three ytterbium ions coordinated to a central [Ru(bpm)3]2+ (bpm = 2,2'-bipyrimidine) motif. Irradiation into the absorption band of the peripheral ytterbium ions at 980 nm engenders emission of the 3MLCT state of the central [Ru(bpm)3]2+ core at 636 nm, which represents the first example of f → d molecular upconversion (UC). Time-resolved measurements reveal a slow rise of the UC emission, which was modeled with a mathematical treatment of the observed kinetics according to a cooperative photosensitization mechanism using a virtual Yb centered doubly excited state followed by energy transfer to the Ru centered 1MLCT state.

Structural basis for DNA recognition and allosteric control of the retinoic acid receptors RAR-RXR.

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.

Molecular Upconversion in Water in Heteropolynuclear Supramolecular Tb/Yb Assemblies.

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.

Time-dependent luminescence loss for individual upconversion nanoparticles upon dilution in aqueous solution.

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.

Quantitative assessment of energy transfer in upconverting nanoparticles grafted with organic dyes.

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.

FRET-based assay to screen inhibitors of HIV-1 reverse transcriptase and nucleocapsid protein.

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.

Large parallelization of STED nanoscopy using optical lattices.

As a scanning microscope, STimulated Emission Depletion (STED) nanoscopy needs parallelization for fast wide-field imaging. Using well-designed optical lattices for depletion together with wide-field excitation and a fast camera for detection, we achieve large parallelization of STED nanoscopy. Wide field of view super-resolved images are acquired by scanning over a single unit cell of the optical lattice, which can be as small as 290 nm * 290 nm. Optical Lattice STED (OL-STED) imaging is demonstrated with a resolution down to 70 nm at 12.5 frames per second.

Site-selective probing of cTAR destabilization highlights the necessary plasticity of the HIV-1 nucleocapsid protein to chaperone the first strand transfer.

The HIV-1 nucleocapsid protein (NCp7) is a nucleic acid chaperone required during reverse transcription. During the first strand transfer, NCp7 is thought to destabilize cTAR, the (-)DNA copy of the TAR RNA hairpin, and subsequently direct the TAR/cTAR annealing through the zipping of their destabilized stem ends. To further characterize the destabilizing activity of NCp7, we locally probe the structure and dynamics of cTAR by steady-state and time resolved fluorescence spectroscopy. NC(11-55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem. This destabilization is achieved through zinc-finger-dependent binding of NC to the G(10) and G(50) residues. Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G(10) and G(50) the appropriate mobility and accessibility for specific recognition by NC. Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway.

Long vs. short-range orders in random subwavelength hole arrays.

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.