Publications by authors named "Antigoni Alexandrou"

27 Publications

  • Page 1 of 1

The tetraspanin CD9 controls migration and proliferation of parietal epithelial cells and glomerular disease progression.

Nat Commun 2019 07 24;10(1):3303. Epub 2019 Jul 24.

Institut National de la Santé et de la Recherche Médicale (Inserm), Unit 970, Paris Cardiovascular Center - PARCC, 56 rue Leblanc, F-75015, Paris, France.

The mechanisms driving the development of extracapillary lesions in focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CGN) remain poorly understood. A key question is how parietal epithelial cells (PECs) invade glomerular capillaries, thereby promoting injury and kidney failure. Here we show that expression of the tetraspanin CD9 increases markedly in PECs in mouse models of CGN and FSGS, and in kidneys from individuals diagnosed with these diseases. Cd9 gene targeting in PECs prevents glomerular damage in CGN and FSGS mouse models. Mechanistically, CD9 deficiency prevents the oriented migration of PECs into the glomerular tuft and their acquisition of CD44 and β1 integrin expression. These findings highlight a critical role for de novo expression of CD9 as a common pathogenic switch driving the PEC phenotype in CGN and FSGS, while offering a potential therapeutic avenue to treat these conditions.
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http://dx.doi.org/10.1038/s41467-019-11013-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6656772PMC
July 2019

Ultra-wide range field-dependent measurements of the relaxivity of GdEuVO nanoparticle contrast agents using a mechanical sample-shuttling relaxometer.

Sci Rep 2017 03 20;7:44770. Epub 2017 Mar 20.

NIMBE, CEA-CNRS, Université Paris-Saclay, CEA Saclay, 91191 Gif-sur-Yvette Cedex, France.

The current trend for Magnetic Resonance Imaging points towards higher magnetic fields. Even though sensitivity and resolution are increased in stronger fields, T1 contrast is often reduced, and this represents a challenge for contrast agent design. Field-dependent measurements of relaxivity are thus important to characterize contrast agents. At present, the field-dependent curves of relaxivity are usually carried out in the field range of 0 T to 2 T, using fast field cycling relaxometers. Here, we employ a high-speed sample shuttling device to switch the magnetic fields experienced by the nuclei between virtually zero field, and the center of any commercial spectrometer. We apply this approach on rare-earth (mixed Gadolinium-Europium) vanadate nanoparticles, and obtain the dispersion curves from very low magnetic field up to 11.7 T. In contrast to the relaxivity profiles of Gd chelates, commonly used for clinical applications, which display a plateau and then a decrease for increasing magnetic fields, these nanoparticles provide maximum contrast enhancement for magnetic fields around 1-1.5 T. These field-dependent curves are fitted using the so-called Magnetic Particle (MP) model and the extracted parameters discussed as a function of particle size and composition. We finally comment on the new possibilities offered by this approach.
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http://dx.doi.org/10.1038/srep44770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5357940PMC
March 2017

Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease.

Blood 2015 Jun 31;125(24):3805-14. Epub 2015 Mar 31.

Paris Center for Cardiovascular Research, Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche-970, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, and Université Paris Descartes, Sorbonne Paris Cité, Paris, France;

Intravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate cell-free heme in human hemolytic diseases, and that heme-laden MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of heme-laden MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD.
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http://dx.doi.org/10.1182/blood-2014-07-589283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4490297PMC
June 2015

Differential interaction kinetics of a bipolar structure-specific endonuclease with DNA flaps revealed by single-molecule imaging.

PLoS One 2014 20;9(11):e113493. Epub 2014 Nov 20.

Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS (Centre National pour la Recherche Scientifique) UMR (Unité Mixte de Recherche) 7645, Inserm (Institut national de la santé et de la recherche médicale) U696, Palaiseau, France.

As DNA repair enzymes are essential for preserving genome integrity, understanding their substrate interaction dynamics and the regulation of their catalytic mechanisms is crucial. Using single-molecule imaging, we investigated the association and dissociation kinetics of the bipolar endonuclease NucS from Pyrococcus abyssi (Pab) on 5' and 3'-flap structures under various experimental conditions. We show that association of the PabNucS with ssDNA flaps is largely controlled by diffusion in the NucS-DNA energy landscape and does not require a free 5' or 3' extremity. On the other hand, NucS dissociation is independent of the flap length and thus independent of sliding on the single-stranded portion of the flapped DNA substrates. Our kinetic measurements have revealed previously unnoticed asymmetry in dissociation kinetics from these substrates that is markedly modulated by the replication clamp PCNA. We propose that the replication clamp PCNA enhances the cleavage specificity of NucS proteins by accelerating NucS loading at the ssDNA/dsDNA junctions and by minimizing the nuclease interaction time with its DNA substrate. Our data are also consistent with marked reorganization of ssDNA and nuclease domains occurring during NucS catalysis, and indicate that NucS binds its substrate directly at the ssDNA-dsDNA junction and then threads the ssDNA extremity into the catalytic site. The powerful techniques used here for probing the dynamics of DNA-enzyme binding at the single-molecule have provided new insight regarding substrate specificity of NucS nucleases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0113493PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4239081PMC
December 2015

Single YVO4:Eu nanoparticle emission spectra using direct Eu3+ ion excitation with a sum-frequency 465-nm solid-state laser.

Opt Express 2014 Aug;22(17):20542-50

We report emission spectrum measurements on single YxEu(1-x)VO4 nanoparticles. The inhomogeneous widths of the emission peaks are identical for single nanoparticles and for ensembles of nanoparticles, while being broader than those of the bulk material. This indicates that individual nanoparticles are identical in terms of the distribution of different local Eu3+ sites due to crystalline defects and confirms their usability as identical, single-particle oxidant biosensors. Moreover, we report a 465 nm solid-state laser based on sum-frequency mixing that provides a compact, efficient solution for direct Eu3+ excitation of these nanoparticles. Both these two aspects should broaden the scope of Eu-doped nanoparticle applications.
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http://dx.doi.org/10.1364/OE.22.020542DOI Listing
August 2014

[Map the intracellular concentration of reactive oxygen species].

Med Sci (Paris) 2014 Oct 14;30(10):848-50. Epub 2014 Oct 14.

Laboratoire optique et biosciences, CNRS UMR7645, Inserm U696, École polytechnique, route de Saclay, 91128 Palaiseau, France.

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http://dx.doi.org/10.1051/medsci/20143010010DOI Listing
October 2014

Multifunctional rare-Earth vanadate nanoparticles: luminescent labels, oxidant sensors, and MRI contrast agents.

ACS Nano 2014 Nov 20;8(11):11126-37. Epub 2014 Oct 20.

Laboratoire d'Optique et Biosciences, Ecole Polytechnique , CNRS UMR 7645-INSERM U696, 91128 Palaiseau Cedex, France.

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.
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http://dx.doi.org/10.1021/nn504170xDOI Listing
November 2014

Regulation of the ROS response dynamics and organization to PDGF motile stimuli revealed by single nanoparticle imaging.

Chem Biol 2014 May 10;21(5):647-56. Epub 2014 Apr 10.

Laboratoire d'Optique et Biosciences, Ecole Polytechnique Université Paris Saclay, Inserm U696, CNRS 7645, 91128 Palaiseau, France.

Although reactive oxygen species (ROS) are better known for their harmful effects, more recently, H2O2, one of the ROS, was also found to act as a secondary messenger. However, details of spatiotemporal organization of specific signaling pathways that H2O2 is involved in are currently missing. Here, we use single nanoparticle imaging to measure the local H2O2 concentration and reveal regulation of the ROS response dynamics and organization to platelet-derived growth factor (PDGF) signaling. We demonstrate that H2O2 production is controlled by PDGFR kinase activity and EGFR transactivation, requires a persistent stimulation, and is regulated by membrane receptor diffusion. This temporal filtering is impaired in cancer cells, which may determine their pathological migration. H2O2 subcellular mapping reveals that an external PDGF gradient induces an amplification-free asymmetric H2O2 concentration profile. These results support a general model for the control of signal transduction based only on membrane receptor diffusion and second messenger degradation.
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http://dx.doi.org/10.1016/j.chembiol.2014.02.020DOI Listing
May 2014

Calibrating optical tweezers with Bayesian inference.

Opt Express 2013 Dec;21(25):31578-90

We present a new method for calibrating an optical-tweezer setup that does not depend on input parameters and is less affected by systematic errors like drift of the setup. It is based on an inference approach that uses Bayesian probability to infer the diffusion coefficient and the potential felt by a bead trapped in an optical or magnetic trap. It exploits a much larger amount of the information stored in the recorded bead trajectory than standard calibration approaches. We demonstrate that this method outperforms the equipartition method and the power-spectrum method in input information required (bead radius and trajectory length) and in output accuracy.
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http://dx.doi.org/10.1364/OE.21.031578DOI Listing
December 2013

Receptor displacement in the cell membrane by hydrodynamic force amplification through nanoparticles.

Biophys J 2013 Jul;105(1):116-26

Laboratoire d'Optique et Biosciences, Ecole Polytechnique, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale U696, Palaiseau Cedex, France.

We introduce an intrinsically multiplexed and easy to implement method to apply an external force to a biomolecule and thus probe its interaction with a second biomolecule or, more generally, its environment (for example, the cell membrane). We take advantage of the hydrodynamic interaction with a controlled fluid flow within a microfluidic channel to apply a force. By labeling the biomolecule with a nanoparticle that acts as a kite and increases the hydrodynamic interaction with the fluid, the drag induced by convection becomes important. We use this approach to track the motion of single membrane receptors, the Clostridium perfringens ε-toxin (CPεT) receptors that are confined in lipid raft platforms, and probe their interaction with the environment. Under external force, we observe displacements over distances up to 10 times the confining domain diameter due to elastic deformation of a barrier and return to the initial position after the flow is stopped. Receptors can also jump over such barriers. Analysis of the receptor motion characteristics before, during, and after a force is applied via the flow indicates that the receptors are displaced together with their confining raft platform. Experiments before and after incubation with latrunculin B reveal that the barriers are part of the actin cytoskeleton and have an average spring constant of 2.5 ± 0.6 pN/μm before vs. 0.6 ± 0.2 pN/μm after partial actin depolymerization. Our data, in combination with our previous work demonstrating that the ε-toxin receptor confinement is not influenced by the cytoskeleton, imply that it is the raft platform and its constituents rather than the receptor itself that encounters and deforms the barriers formed by the actin cytoskeleton.
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http://dx.doi.org/10.1016/j.bpj.2013.05.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3699754PMC
July 2013

Probing membrane protein interactions with their lipid raft environment using single-molecule tracking and Bayesian inference analysis.

PLoS One 2013 3;8(1):e53073. Epub 2013 Jan 3.

Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS, INSERM U696, Palaiseau, France.

The statistical properties of membrane protein random walks reveal information on the interactions between the proteins and their environments. These interactions can be included in an overdamped Langevin equation framework where they are injected in either or both the friction field and the potential field. Using a Bayesian inference scheme, both the friction and potential fields acting on the ε-toxin receptor in its lipid raft have been measured. Two types of events were used to probe these interactions. First, active events, the removal of cholesterol and sphingolipid molecules, were used to measure the time evolution of confining potentials and diffusion fields. Second, passive rare events, de-confinement of the receptors from one raft and transition to an adjacent one, were used to measure hopping energies. Lipid interactions with the ε-toxin receptor are found to be an essential source of confinement. ε-toxin receptor confinement is due to both the friction and potential field induced by cholesterol and sphingolipids. Finally, the statistics of hopping energies reveal sub-structures of potentials in the rafts, characterized by small hopping energies, and the difference of solubilization energy between the inner and outer raft area, characterized by higher hopping energies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053073PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536804PMC
August 2013

Observing the confinement potential of bacterial pore-forming toxin receptors inside rafts with nonblinking Eu(3+)-doped oxide nanoparticles.

Biophys J 2012 May 15;102(10):2299-308. Epub 2012 May 15.

Laboratoire d'Optique et Biosciences, Ecole Polytechnique, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale U696, Palaiseau, France.

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.
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http://dx.doi.org/10.1016/j.bpj.2012.03.072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3352999PMC
May 2012

A Bayesian inference scheme to extract diffusivity and potential fields from confined single-molecule trajectories.

Biophys J 2012 May 15;102(10):2288-98. Epub 2012 May 15.

Laboratoire d'Optique et Biosciences, Ecole Polytechnique, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale U696, Palaiseau, France.

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.
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http://dx.doi.org/10.1016/j.bpj.2012.01.063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3353014PMC
May 2012

Biological applications of rare-earth based nanoparticles.

ACS Nano 2011 Nov 18;5(11):8488-505. Epub 2011 Oct 18.

Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS UMR7645 INSERM U696, 91128 Palaiseau Cedex, France.

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.
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http://dx.doi.org/10.1021/nn202378bDOI Listing
November 2011

Rails and anchors: guiding and trapping droplet microreactors in two dimensions.

Lab Chip 2011 Mar 9;11(5):813-21. Epub 2010 Nov 9.

Laboratoire d'Hydrodynamique (LadHyX) and Department of Mechanics, Ecole Polytechnique, CNRS, 91128 Palaiseau, France.

This paper presents a method to control the motion of nanolitre drops in a wide and thin microchannel, by etching fine patterns into the channel's top surface. Such control is possible for drops that are squeezed by the channel roof, by allowing them to reduce their surface energy as they enter into a local depression. The resulting gain in surface energy pulls a drop into the groove such that localized holes can be used as anchors for holding drops, while linear patterns can be used as rails to guide them along complex trajectories. An anchored drop can remain stationary indefinitely, as long as the driving flow rate is below a critical value which depends on the hole and drop sizes. By micro-fabricating holes into a grid pattern, drops can be arrayed and held in the observation field of a microscope against the mean carrier flow. Their contents can then be modulated by gas exchange with the flowing carrier oil. We demonstrate in particular how the pH or the oxygen levels within the drops can be controlled spatially and temporally, either by exposing rows of drops to two streams of oil at different gas concentrations or by periodically switching oil inputs to vary the gas concentration of drops as a function of time. Oxygen control is used to selectively deoxygenate droplets that encapsulate red blood cells from patients suffering from sickle cell disease, in order to study the polymerization of intracellular hemoglobin. Cycles of oxygenation and deoxygenation of anchored droplets induce depolymerization and polymerization of the hemoglobin, thus providing a method to simulate the cycling that takes place in physiological flows.
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http://dx.doi.org/10.1039/c0lc00104jDOI Listing
March 2011

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

Lab Chip 2010 Oct 6;10(19):2505-12. Epub 2010 Jul 6.

Laboratoire d'Optique et Biosciences (LOB), Ecole Polytechnique, INSERM U696, CNRS, 91128 Palaiseau, France.

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.
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http://dx.doi.org/10.1039/c004390gDOI Listing
October 2010

Quantifying biomolecule diffusivity using an optimal Bayesian method.

Biophys J 2010 Feb;98(4):596-605

Institut Pasteur, Centre National de la Recherche Scientifique URA 2171, Unit In Silico Genetics, Paris, France.

We propose a Bayesian method to extract the diffusivity of biomolecules evolving freely or inside membrane microdomains. This approach assumes a model of motion for the particle considered, namely free Brownian motion or confined diffusion. In each framework, a systematic Bayesian scheme is provided for estimating the diffusivity. We show that this method reaches the best performances theoretically achievable. Its efficiency overcomes that of widely used methods based on the analysis of the mean-square displacement. The approach presented here also gives direct access to the uncertainty on the estimation of the diffusivity and predicts the number of steps of the trajectory necessary to achieve any desired precision. Its robustness with respect to noise on the position of the biomolecule is also investigated.
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http://dx.doi.org/10.1016/j.bpj.2009.10.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2820641PMC
February 2010

Suppression of perturbed free-induction decay and noise in experimental ultrafast pump-probe data.

Opt Lett 2009 Oct;34(20):3226-8

Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS, France.

We apply a Fourier filtering technique for the global removal of coherent contributions, like perturbed free-induction decay, and noise, to experimental pump-probe spectra. A further filtering scheme gains access to spectra otherwise only recordable by scanning the probe's center frequency with adjustable spectral resolution. These methods cleanse pump-probe data and allow improved visualization and simpler analysis of the contained dynamics. We demonstrate these filters using visible pump/mid-infrared probe spectroscopy of ligand dissociation in carboxyhemoglobin.
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http://dx.doi.org/10.1364/OL.34.003226DOI Listing
October 2009

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

Nat Nanotechnol 2009 Sep 9;4(9):581-5. Epub 2009 Aug 9.

Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS, INSERM U696, Palaiseau, France.

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.
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http://dx.doi.org/10.1038/nnano.2009.200DOI Listing
September 2009

Organic functionalization of luminescent oxide nanoparticles toward their application as biological probes.

Langmuir 2008 Oct 5;24(19):11018-26. Epub 2008 Sep 5.

Laboratoire de Physique de la Matière Condensée, Ecole Polytechnique, CNRS, Route de Saclay, 91128 Palaiseau, France.

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.
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http://dx.doi.org/10.1021/la8015468DOI Listing
October 2008

Counting the number of proteins coupled to single nanoparticles.

J Am Chem Soc 2007 Oct 29;129(42):12592-3. Epub 2007 Sep 29.

Laboratoire d'Optique et Biosciences and Laboratoire de Physique de la Matière Condensée, Ecole Polytechnique, CNRS, 91128 Palaiseau, France, and INSERM U696, 91128, Palaiseau, France.

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http://dx.doi.org/10.1021/ja0731975DOI Listing
October 2007

Direct observation of ligand transfer and bond formation in cytochrome c oxidase by using mid-infrared chirped-pulse upconversion.

Proc Natl Acad Sci U S A 2007 Oct 25;104(40):15705-10. Epub 2007 Sep 25.

Laboratoire d'Optique et Biosciences, Ecole Polytechnique, Centre National de la Recherche Scientifique and Institut National de la Santé et de la Recherche Médicale U696, 91128 Palaiseau, France.

We have implemented the recently demonstrated technique of chirped-pulse upconversion of midinfrared femtosecond pulses into the visible in a visible pump-midinfrared probe experiment for high-resolution, high-sensitivity measurements over a broad spectral range. We have succeeded in time-resolving the CO ligand transfer process from the heme Fe to the neighboring Cu(B) atom in the bimetallic active site of mammalian cytochrome c oxidase, which was known to proceed in <1 ps, using the full CO vibrational signature of Fe-CO bond breaking and Cu(B)-CO bond formation. Our differential transmission results show a delayed onset of the appearance of the Cu(B)-bound species (200 fs), followed by a 450-fs exponential rise. Trajectories calculated by using molecular-dynamics simulations with a Morse potential for the Cu(B)-C interaction display a similar behavior. Both experimental and calculated data strongly suggest a ballistic contribution to the transfer process.
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http://dx.doi.org/10.1073/pnas.0703279104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2000433PMC
October 2007

Single lanthanide-doped oxide nanoparticles as donors in fluorescence resonance energy transfer experiments.

J Phys Chem B 2006 Oct;110(39):19264-70

Laboratory for Optics and Biosciences, CNRS UMR7645, INSERM U696, Ecole Polytechnique, F-91128 Palaiseau Cedex, France.

We used lanthanide-ion doped oxide nanoparticles, Y(0.6)Eu(0.4)VO(4), as donors in fluorescent resonance energy transfer (FRET) experiments. The choice of these nanoparticles allows us to combine the advantages of the lanthanide-ion emission, in particular the long lifetime and the large Stokes shift between absorption and emission, with the detectability of the nanoparticles at the single-particle level. Using cyanine 5 (Cy5) organic molecules as acceptors, we demonstrated FRET down to the single-nanoparticle level. We showed that, due to the long donor lifetime, unambiguous and precise FRET measurements can be performed in solution even in the presence of large free acceptor concentrations. Highly efficient energy transfer was obtained for a large number of acceptor molecules per donor nanoparticle. We determined FRET efficiencies as a function of Cy5 concentration which are in good agreement with a multiple acceptor-multiple donor calculation. On the basis of the donor emission recovery due to acceptor photobleaching, we demonstrated energy transfer from single-nanoparticle donors in fluorescence microscopy experiments.
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http://dx.doi.org/10.1021/jp063229vDOI Listing
October 2006

Fourier-transform coherent anti-Stokes Raman scattering microscopy.

Opt Lett 2006 Feb;31(4):480-2

Laboratoire d'Optique et Biosciences, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7645, Institut National de la Santé et de la Recherche Médicale U696, Ecole Polytechnique, 91128 Palaiseau, France.

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.
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http://dx.doi.org/10.1364/ol.31.000480DOI Listing
February 2006

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

Opt Lett 2005 Apr;30(8):911-3

Laboratoire d'Optique et Biosciences, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale U696, Ecole Polytechnique, 91128 Palaiseau Cedex, France.

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.
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http://dx.doi.org/10.1364/ol.30.000911DOI Listing
April 2005

Coherent vibrational climbing in carboxyhemoglobin.

Proc Natl Acad Sci U S A 2004 Sep 19;101(36):13216-20. Epub 2004 Aug 19.

Centre National de la Recherche Scientifique Unité Mixte de Recherche 7645, Institut National de la Santé et de la Recherche Médicale, Ecole Polytechnique-Ecole Nationale Supérieure de Techniques Avancées, Palaiseau, France.

We demonstrate vibrational climbing in the CO stretch of carboxyhemoglobin pumped by midinfrared chirped ultrashort pulses. By use of spectrally resolved pump-probe measurements, we directly observed the induced absorption lines caused by excited vibrational populations up to v = 6. In some cases, we also observed stimulated emission, providing direct evidence of vibrational population inversion. This study provides important spectroscopic parameters on the CO stretch in the strong-field regime, such as transition frequencies and dephasing times up to the v = 6 to v = 7 vibrational transition. We measured equally spaced vibrational transitions, in agreement with the energy levels of a Morse potential up to v = 6. It is interesting that the integral of the differential absorption spectra was observed to deviate far from zero, in contrast to what one would expect from a simple one-dimensional Morse model assuming a linear dependence of dipole moment with bond length.
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http://dx.doi.org/10.1073/pnas.0401844101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC516550PMC
September 2004