Université PSL

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Single-cell response to stiffness exhibits muscle-like behavior
Laboratoire Physique des biomolécules - D. Mitrossilis, J. Fouchard, A. Guiroy, N. Desprat, N. Rodriguez, B. Fabry and A. Asnacios
Proc. Nat. Acad. Sci. USA - 106(43) :18243–8 - DOI:10.1073/pnas.0903994106 - 2009
Living cells sense the rigidity of their environment and adapt their activity to it. In particular, cells cultured on elastic substrates align their shape and their traction forces along the direction of highest stiffness and preferably migrate towards stiffer regions. Although numerous studies investigated the role of adhesion complexes in rigidity sensing, less is known about the specific contribution of acto-myosin based contractility. Here we used a custom-made single-cell technique to measure the traction force as well as the speed of shortening of isolated myoblasts deflecting microplates of variable stiffness. The rate of force generation increased with increasing stiffness and followed a Hill force–velocity relationship. Hence, cell response to stiffness was similar to muscle adaptation to load, reflecting the force-dependent kinetics of myosin binding to actin. These results reveal an unexpected mechanism of rigidity sensing, whereby the contractile acto-myosin units themselves can act as sensors. This mechanism may translate anisotropy in substrate rigidity into anisotropy in cytoskeletal tension, and could thus coordinate local activity of adhesion complexes and guide cell migration along rigidity gradients.
Coupling DNA unwinding activity with primer synthesis in the bacteriophage T4 primosome
Laboratoire Physique des biomolécules - M. Manosas, M. M. Spiering, Z. Zhuang, S. J. Benkovic and V. Croquette
Nature Chemical Biology - 5(12) :904–12 - DOI:10.1038/nchembio.236 - 2009
The unwinding and priming activities of the bacteriophage T4 primosome, which consists of a hexameric helicase (gp41) translocating 5' to 3' and an oligomeric primase (gp61) synthesizing primers 5' to 3', have been investigated on DNA hairpins manipulated by a magnetic trap. We find that the T4 primosome continuously unwinds the DNA duplex while allowing for primer synthesis through a primosome disassembly mechanism or a new DNA looping mechanism. A fused gp61-gp41 primosome unwinds and primes DNA exclusively via the DNA looping mechanism. Other proteins within the replisome control the partitioning of these two mechanisms by disfavoring primosome disassembly, thereby increasing primase processivity. In contrast to T4, priming in bacteriophage T7 and Escherichia coli involves discrete pausing of the primosome and dissociation of the primase from the helicase, respectively. Thus nature appears to use several strategies to couple the disparate helicase and primase activities within primosomes.
Wetting and spreading
Laboratoire Procédés - Plasmas - Microsystèmes - Bonn D., Eggers J., Iindekeu J., Meunier J., Rolley E.
REVIEWS OF MODERN PHYSICS - 81(2) :739-805 - DOI:10.1103/RevModPhys.81.739 - 2009
Wetting phenomena are ubiquitous in nature and technology. A solid substrate exposed to the environment is almost invariably covered by a layer of fluid material. In this review, the surface forces that lead to wetting are considered, and the equilibrium surface coverage of a substrate in contact with a drop of liquid. Depending on the nature of the surface forces involved, different scenarios for wetting phase transitions are possible; recent progress allows us to relate the critical exponents directly to the nature of the surface forces which lead to the different wetting scenarios. Thermal fluctuation effects, which can be greatly enhanced for wetting of geometrically or chemically structured substrates, and are much stronger in colloidal suspensions, modify the adsorption singularities. Macroscopic descriptions and microscopic theories have been developed to understand and predict wetting behavior relevant to microfluidics and nanofluidics applications. Then the dynamics of wetting is examined. A drop, placed on a substrate which it wets, spreads out to form a film. Conversely, a nonwetted substrate previously covered by a film dewets upon an appropriate change of system parameters. The hydrodynamics of both wetting and dewetting is influenced by the presence of the three-phase contact line separating ``wet'' regions from those that are either dry or covered by a microscopic film only. Recent theoretical, experimental, and numerical progress in the description of moving contact line dynamics are reviewed, and its relation to the thermodynamics of wetting is explored. In addition, recent progress on rough surfaces is surveyed. The anchoring of contact lines and contact angle hysteresis are explored resulting from surface inhomogeneities. Further, new ways to mold wetting characteristics according to technological constraints are discussed, for example, the use of patterned surfaces, surfactants, or complex fluids.
Development of oligonucleotide microarray involving plasma polymerized acrylic acid” Thin Solid Films
Laboratoire Procédés - Plasmas - Microsystèmes - R. Jafari , M. Tatoulian, D. Le Clerre, L. Talini, F. Richard, F. Arefi-Khonsari
Thin Solid Films - 517(19) :5763-8 - DOI:10.1016/j.tsf.2009.03.217 - 2009
This paper presents the manufacturing of biochips by using the COOH– derived polymer coating deposited by plasma polymerization of acrylic acid. This technology is based on depositing a thin layer obtained by plasma polymerization of acrylic acid which allows a further covalent immobilization of biomolecules on glass substrates. The plasma power value was optimized to maximize the stability of plasma polymerized acrylic acid (PPAA) coatings in water, which has a very important role for such applications. In order to obtain a covalent immobilization of DNA probes on the PPAA coated surface, the activation protocol of carboxylic function was carried out with the help of N-Hydroxy Succinimide and 1-Ethyl-3-(3-DimethylAminopropyl) Carbodiimide. The efficiency of PPAA coated in microarray applications was compared with two types of commercial slides. Such surfaces have shown very interesting results in terms of relative density of attached DNA probe molecules and signal-to-background ratio measured for target DNA hybridization. Nonspecific DNA bonding measurements showed only a small amount of nonspecific physisorption between the DNA probe and the PPAA-activated surfaces. This work shows that the plasma polymerization technique can be successfully applied to produce a high-quality glass surface for the manufacturing of DNA arrays.
Determination of Cocaine in Human Plasma by Selective Solid-Phase Extraction Using an Aptamer-Based Sorbent
Laboratoire Sciences Analytiques Bioanalytiques et Miniaturisation - B. Madru, F. Chapuis-Hugon, E. Peyrin, V. Pichon
Anal. Chem. - 81(16) :7081-6 - DOI:10.1021/ac9006667 - 2009
A complete characterization is presented of a highly selective solid-phase extraction (SPE) sorbent which exploits the properties of aptamers. An oligosorbent based on aptamers immobilized on a solid support was synthesized and tested for the selective extraction of cocaine from human plasma. Anticocaine aptamers were immobilized to CNBr-activated Sepharose, and an extraction procedure was developed in pure media. Specific retention of cocaine on the oligosorbent was demonstrated, and the capacity of the support was determined. This oligosorbent was then applied to the selective extraction of cocaine from plasma at a concentration of 0.4 mg L(-1), i.e., corresponding to the plasma concentration reached after an intake of a single dose of cocaine. Extraction recovery close to 90% was obtained. Moreover, interfering compounds that perturbed cocaine quantification when using a standard SPE sorbent were not retained on the oligosorbent, thus allowing fast and reliable analyses of plasma samples with an estimated limit of detection of 0.1 microg mL(-1).
Charge-based characterization of nanometric cationic bifunctional maghemite/silica core/shell particles by capillary zone electrophoresis
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - d'Orlye F, Varenne A, Georgelin T, Siaugue JM, Teste B, Descroix S, Gareil P
Electrophoresis - 30(14) :2572-82 - DOI:10.1002/elps.200800835. - 2009
In view of employing functionalized nanoparticles (NPs) in the context of an immunodiagnostic, aminated maghemite/silica core/shell particles were synthesized so as to be further coated with an antibody or an antigen via the amino groups at their surface. Different functionalization rates were obtained by coating these maghemite/silica core/shell particles with 3-(aminopropyl)triethoxysilane and 2-[methoxy(polyethyleneoxy)propyl]-trimethoxysilane at different molar ratios. Adequate analytical performances with CE coupled with UV-visible detection were obtained through semi-permanent capillary coating with didodecyldimethyl-ammonium bromide, thus preventing particle adsorption. First, the influence of experimental conditions such as electric field strength, injected particle amount as well as electrolyte ionic strength and pH, was evaluated. A charge-dependent electrophoretic mobility was evidenced and the separation selectivity was tuned according to electrolyte ionic strength and pH. The best resolutions were obtained at pH 8.0, high ionic strength (ca. 100 mM), and low total particle volume fraction (ca. 0.055%), thus eliminating interference effects between different particle populations in mixtures. A protocol derived from Kaiser's original description was performed for quantitation of the primary amino groups attached onto the NP surface. Thereafter a correlation between particle electrophoretic mobility and the density of amino groups at their surface was established. Eventually, CE proved to be an easy, fast, and reliable method for the determination of NP effective surface charge density.
Droplet-based microfluidic platforms for the encapsulation and screening of mammalian cells and multicellular organisms
Laboratoire Biochimie - J. Clausell-Tormos, D. Lieber, J.-C. Baret, A. El-Harrak, O.J. Miller, L. Frenz, J. Blouwolff, K.J. Humphry, S. Köster, H. Duan, C. Holtze, D.A. Weitz, A.D. Griffiths and C.A. Merten
Chem. Biol. - 15(5) :427-37 - DOI: 10.1016/j.chembiol.2008.04.004 - 2008
High-throughput, cell-based assays require small sample volumes to reduce assay costs and to allow for rapid sample manipulation. However, further miniaturization of conventional microtiter plate technology is problematic due to evaporation and capillary action. To overcome these limitations, we describe droplet-based microfluidic platforms in which cells are grown in aqueous microcompartments separated by an inert perfluorocarbon carrier oil. Synthesis of biocompatible surfactants and identification of gas-permeable storage systems allowed human cells, and even a multicellular organism (C. elegans), to survive and proliferate within the microcompartments for several days. Microcompartments containing single cells could be reinjected into a microfluidic device after incubation to measure expression of a reporter gene. This should open the way for high-throughput, cell-based screening that can use >1000-fold smaller assay volumes and has approximately 500x higher throughput than conventional microtiter plate assays.
Droplet-based microreactors for the synthesis of magnetic iron oxide nanoparticles. Angewandte
Laboratoire Biochimie - L. Frenz, A. El Harrak, M. Pauly, S. Bégin-Colin, A.D. Griffiths and J.-C. Baret
Chimie Int. Ed. - 47(36) :6817-20 - DOI: 10.1002/anie.200801360 - 2008
Microdroplets for nanoparticles: An extremely reliable method to create droplet pairs, based on hydrodynamic coupling of two spatially separated nozzles, has been developed. Droplets containing the reagents for the precipitation of iron oxide are electrocoalesced to synthesize iron oxide nanoparticles in a very fast (millisecond-scale) and reproducible reaction (see picture).
Flourescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity
Laboratoire Biochimie - J.-C. Baret, O.J. Miller, V. Taly, M. Ryckelynck, A. El-Harrak, L. Frenz, C. Rick, M.L. Samuels, J. B. Hutchison, J.J. Agresti, D.R. Link, D.A. Weitz and A.D. Griffiths
Lab. Chip - 9 :1850-8 - DOI: 10.1039/B902504A - 2008
We describe a highly efficient microfluidic fluorescence-activated droplet sorter (FADS) combining many of the advantages of microtitre-plate screening and traditional fluorescence-activated cell sorting (FACS). Single cells are compartmentalized in emulsion droplets, which can be sorted using dielectrophoresis in a fluorescence-activated manner (as in FACS) at rates up to 2000 droplets s−1. To validate the system, mixtures of E. coli cells, expressing either the reporter enzyme β-galactosidase or an inactive variant, were compartmentalized with a fluorogenic substrate and sorted at rates of 300 droplets s−1. The false positive error rate of the sorter at this throughput was <1 in 104 droplets. Analysis of the sorted cells revealed that the primary limit to enrichment was the co-encapsulation of E. coli cells, not sorting errors: a theoretical model based on the Poisson distribution accurately predicted the observed enrichment values using the starting cell density (cells per droplet) and the ratio of active to inactive cells. When the cells were encapsulated at low density ( 1 cell for every 50 droplets), sorting was very efficient and all of the recovered cells were the active strain. In addition, single active droplets were sorted and cells were successfully recovered.
Regulation of dendritic cell migration by CD74, the MHC class II-associated invariant chain
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Faure-André G, Vargas P, Yuseff MI, Heuzé M, Diaz J, Lankar D, Steri V, Manry J, Hugues S, Vascotto F, Boulanger J, Raposo G, Bono MR, Rosemblatt M, Piel M, Lennon-Duménil AM
Science - 322(5908) :1705-10 - DOI:10.1126/science.1159894 - 2008
Dendritic cells (DCs) sample peripheral tissues of the body in search of antigens to present to T cells. This requires two processes, antigen processing and cell motility, originally thought to occur independently. We found that the major histocompatibility complex II-associated invariant chain (Ii or CD74), a known regulator of antigen processing, negatively regulates DC motility in vivo. By using microfabricated channels to mimic the confined environment of peripheral tissues, we found that wild-type DCs alternate between high and low motility, whereas Ii-deficient cells moved in a faster and more uniform manner. The regulation of cell motility by Ii depended on the actin-based motor protein myosin II. Coupling antigen processing and cell motility may enable DCs to more efficiently detect and process antigens within a defined space.
Physical mechanisms redirecting cell polarity and cell shape in fission yeast
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Terenna CR, Makushok T, Velve-Casquillas G, Baigl D, Chen Y, Bornens M, Paoletti A, Piel M, Tran PT
Curr Biol. - 18(22) :1748-53 - DOI:10.1016/j.cub.2008.09.047. - 2008
The cylindrical rod shape of the fission yeast Schizosaccharomyces pombe is organized and maintained by interactions between the microtubule, cell membrane, and actin cytoskeleton [1]. Mutations affecting any components in this pathway lead to bent, branched, or round cells [2]. In this context, the cytoskeleton controls cell polarity and thus dictates cell shape. Here, we use soft-lithography techniques to construct microfluidic channels to control cell shape. We show that when wild-type rod-shaped cells are physically forced to grow in a bent fashion, they will reorganize their cytoskeleton and redirect cell polarity to make new ectopic cell tips. Moreover, when bent or round mutant cells are physically forced to conform to the wild-type rod-shape, they will reverse their mutational phenotypes by reorganizing their cytoskeleton to maintain proper wild-type-like localization of microtubules, cell-membrane proteins, and actin. Our study provides direct evidence that the cytoskeleton controls cell polarity and cell shape and demonstrates that cell shape also controls the organization of the cytoskeleton in a feedback loop. We present a model of the feedback loop to explain how fission yeast maintain a rod shape and how perturbation of specific parameters of the loop can lead to different cell shapes.
Chiral Colloidal Clusters
Laboratoire Colloïdes et Matériaux Divisés - D. Zerrouki, J. Baudry, D. Pine, P. Chaikin, J. Bibette
Nature - 455 :380-2 - DOI:10.1038/nature07237 - 2008
Chirality is an important element of biology, chemistry and physics. Once symmetry is broken and a handedness is established, biochemical pathways are set. In DNA, the double helix arises from the existence of two competing length scales, one set by the distance between monomers in the sugar backbone, and the other set by the stacking of the base pairs1. Here we use a colloidal system to explore a simple forcing route to chiral structures. To do so we have designed magnetic colloids that, depending on both their shape and induced magnetization, self-assemble with controlled helicity. We model the two length scales with asymmetric colloidal dumbbells linked by a magnetic belt at their waist. In the presence of a magnetic field the belts assemble into a chain and the steric constraints imposed by the asymmetric spheres force the chain to coil. We show that if the size ratio between the spheres is large enough, a single helicity is adopted, right or left. The realization of chiral colloidal clusters opens up a new link between colloidal science and chemistry. These colloidal clusters may also find use as mesopolymers, as optical and light-activated structures2, and as models for enantiomeric separation.
Decompressing Emulsion Droplets Favors Coalescence
Laboratoire Colloïdes et Matériaux Divisés - N. Brémond, A.R. Thiam, J. Bibette
Phys. Rev. Lett. - 100(2) :024501 - DOI:10.1103/PhysRevLett.100.024501 - 2008
The destabilization process of an emulsion under flow is investigated in a microfluidic device. The experimental approach enables us to generate a periodic train of droplet pairs, and thus to isolate and analyze the basic step of the destabilization, namely, the coalescence of two droplets which collide. We demonstrate a counterintuitive phenomenon: coalescence occurs during the separation phase and not during the impact. Separation induces the formation of two facing nipples in the contact area that hastens the connection of the interfaces prior to fusion. Moreover, droplet pairs initially stabilized by surfactants can be destabilized by forcing the separation. Finally, we note that the fusion mechanism is responsible for a cascade of coalescence events in a compact system of droplets where the separation is driven by surface tension.
Measuring the kinetics of biomolecular recognition with magnetic colloids
Laboratoire Colloïdes et Matériaux Divisés - L. Cohen-Tannoudji, E. Bertrand, J. Baudry, C. Robic, C. Goubault, M. Pellissier, A. Johner, F. Thalmann, N. K. Lee, C. M. Marques, J. Bibette
Phys. Rev. Lett. - 100(10) :108301 - DOI:10.1103/PhysRevLett.100.108301 - 2008
We introduce a general methodology based on magnetic colloids to study the recognition kinetics of tethered biomolecules. Access to the full kinetics of the reaction is provided by an explicit measure of the time evolution of the reactant densities. Binding between a single ligand and its complementary receptor is here limited by the colloidal rotational diffusion. It occurs within a binding distance that can be extracted by a reaction-diffusion theory that properly accounts for the rotational Brownian dynamics. Our reaction geometry allows us to probe a large diversity of bioadhesive molecules and tethers, thus providing a quantitative guidance for designing more efficient reactive biomimetic surfaces, as required for diagnostic, therapeutic, and tissue engineering techniques.
Microfluidic high-throughput encapsulation and hydrodynamic self-sorting of single cells
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Chabert M and Viovy JL
Proc. Nat. Acad. Sci. USA - 105(9) :3191-6 - DOI:10.1073/pnas.0708321105 - 2008
We present a purely hydrodynamic method for the high-throughput encapsulation of single cells into picoliter droplets, and spontaneous self-sorting of these droplets. Encapsulation uses a cell-triggered Rayleigh-Plateau instability in a flow-focusing geometry, and self-sorting puts to work two extra hydrodynamic mechanisms: lateral drift of deformable objects in a shear flow, and sterically driven dispersion in a compressional flow. Encapsulation and sorting are achieved on-flight in continuous flow at a rate up to 160 cells per second. The whole process is robust and cost-effective, involving no optical or electrical discrimination, active sorting, flow switching, or moving parts. Successful encapsulation and sorting of 70-80% of the injected cell population into drops containing one and only one cell, with <1% contamination by empty droplets, is demonstrated. The system is also applied to the direct encapsulation and sorting of cancerous lymphocytes from a whole blood mixture, yielding individually encapsulated cancer cells with a >10,000-fold enrichment as compared with the initial mix. The method can be implemented in simple “soft lithography” chips, allowing for easy downstream coupling with microfluidic cell biology or molecular biology protocols.
Controlled proteolysis of normal and pathological prion protein in a microfluidic chip
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Le Nel A, Minc N, Smadja C, Slovakova M, Bilkova Z, Peyrin JM, Viovy JL, Taverna M
Lab. Chip - 8(2) :294-301 - DOI:10.1039/b715238h - 2008
A microreactor for proteinase K (PK)-mediated protein digestion was developed as a step towards the elaboration of a fully integrated microdevice for the detection of pathological prion protein (PrP). PK-grafted magnetic beads were immobilized inside a polydimethylsiloxane (PDMS) microchannel using a longitudinal magnetic field parallel to the flow direction and a magnetic field gradient, thereby forming a matrix for enzymatic digestion. This self-organization provided uniform pore sizes, a low flow resistance and a strong reaction efficiency due to a very thin diffusion layer. The microreactor's performance was first evaluated using a model substrate, succinyl-ala-ala-ala-paranitroanilide (SAAAP). Reaction kinetics were typically accelerated a hundred-fold as compared to conventional batch reactions. Reproducibility was around 98% for on-chip experiments. This microsystem was then applied to the digestion of prion protein from brain tissues. Controlled proteolysis could be obtained by varying the on-chip flow rate, while a complete proteolysis of normal protein was achieved in only three minutes. Extracts from normal and pathological brain homogenates were finally compared and strong discrimination between normal and pathological samples was demonstrated.
Microfluidic droplet-based liquid-liquid extraction
Laboratoire Microfluidique MEMS et nanostructures - P. Mary, V. Studer, P. Tabeling
Anal. Chem. - 80(8) :2680-7 - DOI:10.1021/ac800088s - 2008
We study microfluidic systems in which mass exchanges take place between moving water droplets, formed on-chip, and an external phase (octanol). Here, no chemical reaction takes place, and the mass exchanges are driven by a contrast in chemical potential between the dispersed and continuous phases. We analyze the case where the microfluidic droplets, occupying the entire width of the channel, extract a solutefluoresceinfrom the external phase (extraction) and the opposite case, where droplets reject a soluterhodamineinto the external phase (purification). Four flow configurations are investigated, based on straight or zigzag microchannels. Additionally to the experimental work, we performed two-dimensional numerical simulations. In the experiments, we analyze the influence of different parameters on the process (channel dimensions, fluid viscosities, flow rates, drop size, droplet spacing, ...). Several regimes are singled out. In agreement with the mass transfer theory of Young et al. (Young, W.; Pumir, A.; Pomeau, Y. Phys. Fluids A 1989, 1, 462), we find that, after a short transient, the amount of matter transferred across the droplet interface grows as the square root of time and the time it takes for the transfer process to be completed decreases as Pe-2/3, where Pe is the Peclet number based on droplet velocity and radius. The numerical simulation is found in excellent consistency with the experiment. In practice, the transfer time ranges between a fraction and a few seconds, which is much faster than conventional systems.
Nanofluidics in the Debye layer at hydrophilic and hydrophobic surfaces
Laboratoire Microfluidique MEMS et nanostructures - C.I Bouzigues, P. Tabeling, L. Bocquet
Phys. Rev. Lett. - 101(11) :114503 - DOI:10.1103/PhysRevLett.101.114503 - 2008
By using evanescent waves, we study equilibrium and dynamical properties of liquid-solid interfaces in the Debye layer for hydrophilic and hydrophobic surfaces. We measure velocity profiles and nanotracer concentration and diffusion profiles between 20 and 300 nm from the walls in pressure-driven and electro-osmotic flows. We extract electrostatic and zeta potentials and determine hydrodynamic slip lengths with 10 nm accuracy. The spectacular amplification of the zeta potential resulting from hydrodynamic slippage allows us to clarify for the first time the dynamic origin of the zeta potential.
Theoretical study of sequence-dependent nanopore unzipping of DNA
Laboratoire Nanobiophysiques - U. Bockelmann and V. Viasnoff
Biophys. J. - 94(7) :2716–24 - DOI:10.1529/biophysj.107.111732 - 2008
We theoretically investigate the unzipping of DNA electrically driven through a nanometer-size pore. Taking the DNA base sequence explicitly into account, the unpairing and translocation process is described by a biased random walk in a one-dimensional energy landscape determined by the sequential basepair opening. Distributions of translocation times are numerically calculated as a function of applied voltage and temperature. We show that varying these two parameters changes the dynamics from a predominantly diffusive behavior to a dynamics governed by jumps over local energy barriers. The work suggests experimentally studying sequence effects, by comparing the average value and standard deviation of the statistical distribution of translocation times.
Interference and crosstalk in double optical tweezers using a single laser source
Laboratoire Nanobiophysiques - P. Mangeol and U. Bockelmann
Rev Sci Instrum - 79(8) :083103 - DOI:10.1063/1.2957652 - 2008
Experimental studies of single molecule mechanics require high force sensitivity and low drift, which can be achieved with optical tweezers. We built an optical tweezer setup for force measurements in a two bead assay. A cw infrared laser beam is split by polarization and focused by a high numerical aperture objective to create two traps. The same laser is used to form both traps and to measure the force by back focal plane interferometry. We show that although the two beams entering the microscope are designed to exhibit orthogonal polarization, interference and a significant parasitic force signal occur. Comparing the experimental results with a ray optics model, we show that the interference patterns are caused by the rotation of polarization on microscope lens surfaces and slides. The model qualitatively describes the pattern and the dependence of the parasitic force signal on the experimental parameters. We present two different approaches to experimentally reduce the crosstalk, namely, polarization rectification and frequency shifting.

638 publications.