Antigoni Alexandrou

Biological Applications of Rare-Earth Based Nanoparticles

Biomedicine and cell and molecular biology require powerful imaging techniques of the single molecule scale to the whole organism, either for fundamental science or diagnosis. These applications are however often limited by the optical properties of the available probes. Moreover, in cell biology, the measurement of the cell response with spatial and temporal resolution is a central instrumental problem. This has been one of the main motivations for the development of new probes and imaging techniques either for biomolecule labeling or detection of an intracellular signaling species. The weak photostability of genetically encoded probes or organic dyes has motivated the interest for different types of nanoparticles for imaging such as quantum dots, nanodiamonds, dye-doped silica particles, or metallic nanoparticles. One of the most active fields of research in the past decade has thus been the development of rare-earth based nanoparticles, whose optical properties and low cytotoxicity are promising for biological applications. Attractive properties of rare-earth based nanoparticles include high photostability, absence of blinking, extremely narrow emission lines, large Stokes shifts, long lifetimes that can be exploited for retarded detection schemes, and facile functionalization strategies. The use of specific ions in their compositions can be moreover exploited for oxidant detection or for implementing potent contrast agents for magnetic resonance imaging. In this review, we present these different applications of rare-earth nanoparticles for biomolecule detection and imaging in vitro, in living cells or in small animals. We highlight how chemical composition tuning and surface functionalization lead to specific properties, which can be used for different imaging modalities. We discuss their performances for imaging in comparison with other probes and to what extent they could constitute a central tool in the future of molecular and cell biology.

Fourier-transform coherent anti-Stokes Raman scattering microscopy

We report a novel Fourier-transform-based implementation of coherent anti-Stokes Raman scattering (CARS) microscopy. The method employs a single femtosecond laser source and a Michelson interferometer to create two pulse replicas that are fed into a scanning multiphoton microscope. By varying the time delay between the pulses, we time-resolve the CARS signal, permitting easy removal of the nonresonant background while providing high resolution, spectrally resolved images of CARS modes over the laser bandwidth (approximately 1500 cm(-1)). We demonstrate the method by imaging polystyrene beads in solvent.

Single europium-doped nanoparticles measure temporal pattern of reactive oxygen species production inside cells

Low concentrations of reactive oxygen species, notably hydrogen peroxide (H(2)O(2)), mediate various signalling processes in the cell. Production of these signals is highly regulated and a suitable probe is needed to measure these events. Here, we show that a probe based on a single nanoparticle can quantitatively measure transient H(2)O(2) generation in living cells. The Y(0.6)Eu(0.4)VO(4) nanoparticles undergo photoreduction under laser irradiation but re-oxidize in the presence of oxidants, leading to a recovery in luminescence. Our probe can be regenerated and reliably detects intracellular H(2)O(2) with a 30-s temporal resolution and a dynamic range of 1-45 microM. The differences in the timing of intracellular H(2)O(2) production triggered by different signals were also measured using these nanoparticles. Although the probe is not selective towards H(2)O(2), in many signalling processes H(2)O(2) is, however, the dominant oxidant. In conjunction with appropriate controls, this probe is a powerful tool for unravelling pathways that involve reactive oxygen species.

Organic Functionalization of Luminescent Oxide Nanoparticles toward Their Application As Biological Probes

Luminescent inorganic nanoparticles are now widely studied for their applications as biological probes for in vitro or in vivo experiments. The functionalization of the particles is a key step toward these applications, since it determines the control of the coupling between the particles and the biological species of interest. This paper is devoted to the case of rare earth doped oxide nanoparticles and their functionalization through their surface encapsulation with a functional polysiloxane shell. The first step of the process is the adsorption of silicate ions that will act as a primary layer for the further surface polymerization of the silane, either aminopropyltriethoxysilane (APTES) or glycidoxypropyltrimethoxysilane (GPTMS). The amino- or epoxy- functions born by the silane allow the versatile coupling of the particles with bio-organic species following the chemistry that is commonly used in biochips. Special attention is paid to the careful characterization of each step of the functionalization process, especially concerning the average number of organic functions that are available for the final coupling of the particles with proteins. The surface density of amino or epoxy functions was found to be 0.4 and 1.9 functions per square nanometer for GPTMS and APTES silanized particles, respectively. An example of application of the amino-functionalized particles is given for the coupling with alpha-bungarotoxins. The average number (up to 8) and the distribution of the number of proteins per particle are given, showing the potentialities of the functionalization process for the labeling of biological species.

Multifunctional Rare-Earth Vanadate Nanoparticles: Luminescent Labels, Oxidant Sensors, and MRI Contrast Agents

Collecting information on multiple pathophysiological parameters is essential for understanding complex pathologies, especially given the large interindividual variability. We report here multifunctional nanoparticles which are luminescent probes, oxidant sensors, and contrast agents in magnetic resonance imaging (MRI). Eu(3+) ions in an yttrium vanadate matrix have been demonstrated to emit strong, nonblinking, and stable luminescence. Time- and space-resolved optical oxidant detection is feasible after reversible photoreduction of Eu(3+) to Eu(2+) and reoxidation by oxidants, such as H2O2, leading to a modulation of the luminescence emission. The incorporation of paramagnetic Gd(3+) confers in addition proton relaxation enhancing properties to the system. We synthesized and characterized nanoparticles of either 5 or 30 nm diameter with compositions of GdVO4 and Gd0.6Eu0.4VO4. These particles retain the luminescence and oxidant detection properties of YVO4:Eu. Moreover, the proton relaxivity of GdVO4 and Gd0.6Eu0.4VO4 nanoparticles of 5 nm diameter is higher than that of the commercial Gd(3+) chelate compound Dotarem at 20 MHz. Nuclear magnetic resonance dispersion spectroscopy showed a relaxivity increase above 10 MHz. Complexometric titration indicated that rare-earth leaching is negligible. The 5 nm nanoparticles injected in mice were observed with MRI to concentrate in the liver and the bladder after 30 min. Thus, these multifunctional rare-earth vanadate nanoparticles pave the way for simultaneous optical and magnetic resonance detection, in particular, for in vivo localization evolution and reactive oxygen species detection in a broad range of physiological and pathophysiological conditions.

Fourier transform measurement of two-photon excitation spectra: applications to microscopy and optimal control.

We report a novel Fourier transform method for measuring two-photon excitation spectra. We demonstrate this method using simple dye molecules and discuss its applications in two-photon fluorescence microscopy and optimal control. This method facilitates an intuitive interpretation of recent control experiments in terms of tuning the nonlinear spectrum of the exciting laser source.

A Bayesian Inference Scheme to Extract Diffusivity and Potential Fields from Confined Single-Molecule Trajectories

Currently used techniques for the analysis of single-molecule trajectories only exploit a small part of the available information stored in the data. Here, we apply a Bayesian inference scheme to trajectories of confined receptors that are targeted by pore-forming toxins to extract the two-dimensional confining potential that restricts the motion of the receptor. The receptor motion is modeled by the overdamped Langevin equation of motion. The method uses most of the information stored in the trajectory and converges quickly onto inferred values, while providing the uncertainty on the determined values. The inference is performed on the polynomial development of the potential and on the diffusivities that have been discretized on a mesh. Numerical simulations are used to test the scheme and quantify the convergence toward the input values for forces, potential, and diffusivity. Furthermore, we show that the technique outperforms the classical mean-square-displacement technique when forces act on confined molecules because the typical mean-square-displacement analysis does not account for them. We also show that the inferred potential better represents input potentials than the potential extracted from the position distribution based on Boltzmann statistics that assumes statistical equilibrium.

Sickling of red blood cells through rapid oxygen exchange in microfluidic drops

We have developed a microfluidic approach to study the sickling of red blood cells associated with sickle cell anemia by rapidly varying the oxygen partial pressure within flowing microdroplets. By using the perfluorinated carrier oil as a sink or source of oxygen, the oxygen level within the water droplets quickly equilibrates through exchange with the surrounding oil. This provides control over the oxygen partial pressure within an aqueous drop ranging from 1 kPa to ambient partial pressure, i.e. 21 kPa. The dynamics of the oxygen exchange is characterized through fluorescence lifetime measurements of a ruthenium compound dissolved in the aqueous phase. The gas exchange is shown to occur primarily during and directly after droplet formation, in 0.1 to 0.5 s depending on the droplet diameter and speed. The controlled deoxygenation is used to trigger the polymerization of hemoglobin within sickle red blood cells, encapsulated in drops. This process is observed using polarization microscopy, which yields a robust criterion to detect polymerization based on transmitted light intensity through crossed polarizers.

Inferring maps of forces inside cell membrane microdomains.

Mapping of the forces on biomolecules in cell membranes has spurred the development of effective labels, e.g., organic fluorophores and nanoparticles, to track trajectories of single biomolecules. Standard methods use particular statistics, namely the mean square displacement, to analyze the underlying dynamics. Here, we introduce general inference methods to fully exploit information in the experimental trajectories, providing sharp estimates of the forces and the diffusion coefficients in membrane microdomains. Rapid and reliable convergence of the inference scheme is demonstrated on trajectories generated numerically. The method is then applied to infer forces and potentials acting on the receptor of the toxin labeled by lanthanide-ion nanoparticles. Our scheme is applicable to any labeled biomolecule and results show its general relevance for membrane compartmentation.

Observing the Confinement Potential of Bacterial Pore-Forming Toxin Receptors Inside Rafts with Nonblinking Eu3+-Doped Oxide Nanoparticles

We track single toxin receptors on the apical cell membrane of MDCK cells with Eu-doped oxide nanoparticles coupled to two toxins of the pore-forming toxin family: α-toxin of Clostridium septicum and ε-toxin of Clostridium perfringens. These nonblinking and photostable labels do not perturb the motion of the toxin receptors and yield long uninterrupted trajectories with mean localization precision of 30 nm for acquisition times of 51.3 ms. We were thus able to study the toxin-cell interaction at the single-molecule level. Toxins bind to receptors that are confined within zones of mean area 0.40 ± 0.05 μm(2). Assuming that the receptors move according to the Langevin equation of motion and using Bayesian inference, we determined mean diffusion coefficients of 0.16 ± 0.01 μm(2)/s for both toxin receptors. Moreover, application of this approach revealed a force field within the domain generated by a springlike confining potential. Both toxin receptors were found to experience forces characterized by a mean spring constant of 0.30 ± 0.03 pN/μm at 37°C. Furthermore, both toxin receptors showed similar distributions of diffusion coefficient, domain area, and spring constant. Control experiments before and after incubation with cholesterol oxidase and sphingomyelinase show that these two enzymes disrupt the confinement domains and lead to quasi-free motion of the toxin receptors. Our control data showing cholesterol and sphingomyelin dependence as well as independence of actin depolymerization and microtubule disruption lead us to attribute the confinement of both receptors to lipid rafts. These toxins require oligomerization to develop their toxic activity. The confined nature of the toxin receptors leads to a local enhancement of the toxin monomer concentration and may thus explain the virulence of this toxin family.