Holographic Traction Force Microscopy

Traction Force Microscopy (TFM) computes the forces exerted at the surface of an elastic material by measuring induced deformations in volume. It is used to determine the pattern of the adhesion forces exerted by cells or by cellular assemblies grown onto a soft deformable substrate. Typically, colloidal particles are dispersed in the substrate and their displacement is monitored by fluorescent microscopy. As with any other fluorescent techniques, the accuracy in measuring a particule’s position is ultimately limited by the number of evaluated fluorescent photons. Here, we present a TFM technique based on the detection of probe particle displacements by holographic tracking microscopy. We show that nanometer scale resolutions of the particle displacements can be obtained and determine the maximum volume fraction of markers in the substrate. We demonstrate the feasibility of the technique experimentally and measure the three-dimensional force fields exerted by colorectal cancer cells cultivated onto a polyacrylamide gel substrate

Suspensions colloïdales concentrées sous écoulement (L importance des contacts)

Nous avons étudié dans notre thèse la microstructure d'une suspension colloïdale concentrée lors de son écoulement. En effet, si la rhéologie des suspensions diluées est bien comprise, le comportement des suspensions concentrées est beaucoup plus difficile à décrire. On sait qu'à faibles vitesses de déformation, si la suspension est suffisamment monodisperse, la suspension est rhéofluidifiante. À plus forts taux de cisaillement, la suspension est rhéoépaississante. L'une des hypothèses permettant de comprendre ce comportement est la formation de clusters hydrodynamiques, au sein desquels les particules sont maintenues par les forces de lubrification, dont les contacts entre les particules gouvernent le passage vers un état rhéoépaississant. Dans une première partie, nous avons étudié par simulation numérique (DPD) les contacts entre particules et l'organisation spatiale des contraintes entre particules au contact. Une deuxième partie, expérimentale, est consacrée à la localisation, par microscopie de fluorescence, des contacts entre particules voisines. Les simulations numériques nous ont montré que le champ de contrainte interparticulaire était en effet fortement anisotrope, mais, contrairement à ce que laissent penser les hypothèses de clusters hydrodynamiques ou les chaînes des forces, cette anisotropie a son axe préférentiel dans un plan parallèle à la direction de l'écoulement. Les mesures expérimentales nous ont seulement permis d'imager les particules les plus proches au contact, en l'absence d'écoulement. Nous avons montré que le FRET permettait d'observer les contacts entre particules colloïdales de taille micronique.We have studied the microstructure of concentrated colloidal suspension under flow. Although the rheology of dilute suspensions is well understood, the behavior of concentrated suspensions is much more difficult to describe. In monodisperse solutions, the rheological behavior of the suspension is shear-thinning. At stronger shear rates, the suspension shear thickens. One of the hypotheses allowing understanding this behavior is the formation of hydrodynamic c1usters, inside which partic1es are maintained by the lubrification forces. We sought to test this hypothesis by following the stress between partic1es with two tools: 1- Numerical simulation by Dissipative Partic1e Dynamics (DPD) 2- Experimental measures of fluorescence transfer between neighbouring partic1es in the suspension. The simulation shows that, although partic1e to not exhibit layering along the flow, the interparticular stress pattern is strongly anisotropie. The propagation of stress no longer occurs in the compression quadrant of the deformation, but in a plane paralle1 to the flow direction. In this regime, of small interpartic1es distances, the hydrodynamic stress between adjacent partic1es dominates the rheological response. Experimental measurements show that the FRET allows us to observe the contacts between colloidal micro-partic1es. However, we could not use this technique to visualize a network of contacts in a suspension under flow for several reasons: - The maximum distance observable by FRET is too low and only a small fraction of colloids in contact is actually observed. - Acquisition time of an image is much too long for it to be taken under flow.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Suspensions colloïdales concentrées sous contrainte (instrumentation multispeckle, théorie et expériences)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

CNRS School ``Nanophysics for Health'', 5-9 November 2012, Mittelwhir, France PREFACE

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Ciment à comportement rhéologique ajustable

Actuellement, les industries cimentières utilisent des polymères, appelés superplastifiants, pour faciliter la mise en place des pâtes de ciment. Cependant, ces suspensions, en présence de superplastifiants, deviennent souvent instables et sédimentent. Le but de ce projet était donc de trouver un nouveau polymère permettant de contrôler la rhéologie des pâtes de ciment. Notre choix s est porté sur des polymères thermoassociatifs qui présentent des caractéristiques rhéologiques changeantes avec la température. Nous étudierons le comportement rhéologique de ces systèmes thermoassociatifs et nous montrerons comment ces polymères influent sur les propriétés rhéologiques des pâtes de ciment et de deux systèmes modèles (quartz et silice monodisperse). Enfin, nous corrèlerons ces résultats avec des mesures d adsorption du polymère sur les particules de ciment.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Etude expérimentale de cinétique de deux systèmes (complexation de l'ADN par la protéine NCp7 et dynamique d'une suspension colloïdale vitreuse)

Dans la première partie de cette thèse, nous étudions la cinétique de la complexation d'un double brin d'ADN par la protéine NCp7. Pour ce faire, nous étudions l'évolution des propriétés mécaniques de l'ADN au fur et à mesure de sa complexation, en étirant la complexe ADN/NCp7 à l'aide d'un montage de piégeage optique. Nous avons observé que la longueur de persistance du complexe diminue au fur et à mesure de la complexation. En utilisant un modèle statistique décrivant l'évolution de la flexibilité de l'ADN complexé par NCp7. Notre principal résultat est que la fraction//phi de paires de bases ayant réagi n'est pas une fonction linéaire du temps aux faibles //phi. Nous interprétons nos résultats en supposant que l'adsorption de NCp7 sur l'ADN est fortement coopérative. Dans deuxième chapitre, nous décrivons la dynamique de particules sondes dans une suspension vitreuse colloïdale de Laponite. La Laponite est une particule colloïdale discoïdale de 25nm de diamètre et de 0.92 nm d'épaisseur. Nous utilisons une expérience de microscopie en onde évanescente, et suivons le mouvement de particules fluorescentes de latex. Nous imageons ensuite ces particules. Nous montrons que, pour un mouvement possédant une seule échelle de temps caractéristique, elle est simplement une fonction linéaire du temps. Nous obtenons que, quelle que soit leur taille, le mouvement des particules sondes peut être décrit par une succession de deux modes dynamiques, où le mode le plus rapide correspond à la diffusion des particules dans un fluide viscoélastique.In the first part of this thesis, we study the kinetics of the complexation of a double-stranded DNA byNCp7 protein. To do this, we study the evolution of mechanical properties of DNA and its complexation by stretching the DNA/NCp7 complex with a optical trap. We observed that the persistence length of the complex decreases progressively during the complexation. Using astatistical model we describe the evolution of the flexibility of DNA complexed with NCp7. Our main result is that the fraction phi of base pairs that have reacted is not a linear function of time at low phi.We interpret our results assuming that the adsorption of NCp7 on DNA is highly cooperative. In the second chapter, we describe the dynamics of probe particles in a colloidal glassy suspension of Laponite. Laponite is a colloidal discoidal particle of 25 nm in diameter and 0.92 nm thick. We take advantage of evanescent wave microscopy, and follow the movement of fluorescent latex particles.Then we image these particles. We show that for a movement that has a single characteristic time scale, it is simply a linear function of time. We find that, what ever their size, the motion of probe particles can be described by a succession of two dynamic modes, where the fastest mode corresponds to the diffusion of particles in a viscoelastic fluid.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF

Viscoelastic Properties of Poly(vinyl alcohol) Hydrogels Having Permanent and Transient Cross-Links Studied by Microrheology, Classical Rheometry, and Dynamic Light Scattering

International audienceDynamics of poly(vinyl alcohol) (PVA) hydrogels having chemical and physical transient cross-links simultaneously (dual cross-link PVA gels) were studied by microrheology based on diffusing wave spectroscopy (DWS), classical rheology and single dynamic light scattering (DLS), and compared with those of corresponding chemical and physical PVA gels. Three different relaxation modes (fast, intermediate and slow modes) are observed for physical gels, while one mode (fast mode) is found for chemical gels, and two (fast and intermediate) for dual cross-link gels. The three modes are attributed respectively to Brownian diffusion of PVA polymer or collective diffusion of the network or gel mode (fast mode), macroscopic stress relaxation (intermediate mode whose characteristic time shows q(0) dependence) and Brownian diffusion of aggregates (slow mode). Microrheological measurements are in good agreement with macrorheological showing segmental Rouse mode dynamics in the high frequency range. For physical gels, we found Maxwell type viscoelasticity characterized by a crossover frequency (maximum of G '') and G' similar to omega(2) and G '' similar to omega(1) in the lower frequency range. The chemical gels displayed an elastic plateau with low G '' at low frequency. For the dual cross-link gel a maximum of G '' was observed, and its characteristic time agrees with that of the intermediate mode measured by DLS. We show that this relaxation mode corresponds to the associative Rouse mode characterized by G' = G '' similar to omega(0.5), depending on the dissociation rate of the reversible transient cross links We propose a stress relaxation mechanism of the PVA chains in the presence of elastically inactive but associative transient cross links which induces incomplete stress relaxation

Spectral properties of polypyridyl ruthenium complexes intercalated in DNA: theoretical insights into the surrounding effects of [Ru(dppz)(bpy)2]2+

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Holographic Traction Force Microscopy

International audienceTraction Force Microscopy (TFM) computes the forces exerted at the surface of an elastic material by measuring induced deformations in volume. It is used to determine the pattern of the adhesion forces exerted by cells or by cellular assemblies grown onto a soft deformable substrate. Typically, colloidal particles are dispersed in the substrate and their displacement is monitored by fluorescent microscopy. As with any other fluorescent techniques, the accuracy in measuring a particule's position is ultimately limited by the number of evaluated fluorescent photons. Here, we present a TFM technique based on the detection of probe particle displacements by holographic tracking microscopy. We show that nanometer scale resolutions of the particle displacements can be obtained and determine the maximum volume fraction of markers in the substrate. We demonstrate the feasibility of the technique experimentally and measure the three-dimensional force fields exerted by colorectal cancer cells cultivated onto a polyacrylamide gel substrate

Formation and Self-Organization Kinetics of alpha-CD/PEO-Based Pseudo-Polyrotaxanes in Water. A Specific Behavior at 30 degrees C

alpha-Cyclodextrins (alpha-CDs) have the ability to form inclusion complexes with poly(ethylene oxide) (PEO) polymer chains. These pseudo-polyrotaxanes (PPRs) can be obtained by quenching an alpha-CD/PEO mixture in water from 70 degrees C down to a lower temperature (typically in the range from 5 to 30 degrees C) thanks to favorable interactions between alpha-CD cavities and PEO chains. Moreover, starting from a liquid alpha-CD/PEO mixture at a total mass fraction of 15% w/w at 70 degrees C, the formation of PPRs with time at a lower temperature induces a white physical gel with time, and phase separation is observed. We established that PPR molecules are exclusively found in the precipitated phase although unthreaded alpha-CD molecules and unthreaded PEO chains are in the liquid phase. At 30 degrees C, the physical gel formation is much slower than at 5 degrees C. At 30 degrees C, we established that, in a first step, alpha-CDs thread onto PEO chains, forming PPR molecules which are not in good solvent conditions in water. At a higher length scale, rapid aggregation of the PPR molecules occurs, and threaded alpha-CD-based nanocylinders form (cylinder length L 5.7 nm and cylinder radius R = 4.7 nm). At a higher length scale, alpha-CD-based nanocylinders associate in a Gaussian way, engendering the formation of precipitated domains which are responsible for the high turbidity of the studied system. At the end of this first step (i.e., after 20 min), the system still remains liquid and the PPRs are totally formed. Then, in a second step (i.e., after 150 min), the system undergoes its reorganization characterized by a compacity increase of the precipitated domains and forms a physical gel. We found that PPRs are totally formed after 20 min at 30 degrees C and that the system stays in a nongel state up to 150 min. This opens new perspectives regarding the PPR chemical modification: between these two characteristic times, we can easily envisage an efficient chemical modification of the PPR molecules in water, as for instance,in end-capping reaction leading to the synthesis of polyrotaxanes