Nanometer-Scale Resolution Achieved with Nonradiative Excitation

International audienceNonradiative Excitation Fluorescence Microscopy (NEFM) is a promising technique allowing the observation of biological samples beyond the diraction limit. By coating a substrate with an homogeneous monolayer of quantum dots (QDs), NEFM is achieved through a nonradiative energy transfer from QDs (donors) to dye molecules located in the sample (acceptors). The excitation depth of the sample is then given by the För-ster radius, which corresponds to few nanometers above the surface. The powerful axial resolution of NEFM is highlighted by observing the adhesion of Giant Unilamellar Vesi-cles (GUVs) on strong interaction with coated surfaces. In this paper, we demonstrate that the QD-quenching level is valuable to calculate and map the distance between the membrane and the surface with a high precision along the optical axis. By tuning the electrostatic interactions between the membrane and the substrate, we have been able to measure a height displacement of ≈ 1 nm of the lipid membrane. The experimental results were discussed according to simulations, which take into account all the common forces appearing between lipid membranes and surfaces

Microfluidique : des principes physiques au Lab-on-Chip et applications à la Santé

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Dynamique de billes d'agarose et de vésicules géantes en adhésion sous un écoulement de cisaillement.

Adhesion is a fundamental process which influence the release and the behaviour of pathologist processes, such like circulating cells on blood vessels (inflammatory response). His understanding need knowledge of biological mechanisms but also a physical description of the movement of deformable objects under a shear flow, interacted with a wall. Our work use model systems (vesicles, beads) to identify the role of adhesion parameters (density, mobility of adhesion molecules and deformability of the object) on the movement.We develop a specific interaction : chelation of one nickel ion by two histidines. Imidazole, the complexant part of the histidine, is used to tune the interaction strenght. We described the movement of membranar lipids under flow when vesicles are under strong adhesion. We show the existence of a surfacic flow which depend of the shape of the vesicle and of the adhesion force. By tuning the specific interaction on beads, we show the specificity of the interaction, original phenomenon of capture, detachment and movement of beads under adhesion and under a shear flow.L'adhésion est un processus fondamental qui influence le déclenchement et le déroulement des processus pathologiques, notamment concernant les cellules circulantes aux parois vasculaires (réaction inflammatoire dans le flux sanguin). Sa compréhention nécessite la connaissance des mécanismes biologiques impliqués mais aussi d'un cadre physique de description du mouvement sous flux d'objets déformables en interaction avec une paroi. Notre travail utilise des systèmes modèles (vésicules, billes) pour identifier le rôle de paramètres de l'adhésion (densité, mobilité des ligands membranaires, déformabilité) sur le mouvement. Le système d'interaction développé est la chélation entre un ion nickel et deux histidines. L'imidazole, partie complexante de l'histidine, va permettre de réguler la force de l'interaction. Nous avons décrit le mouvement des lipides membranaires sous flux dans le cas de vésicules en interaction forte. On a mis en évidence la présence d'un flux surfacique dépendant de la forme de l'objet et de la force de l'adhésion. En régulant l'adhésion spécifique faible de billes, nous montrons la spécificité de l'interaction, des phénomènes originaux de capture, de détachement et de déplacement en adhésion sous écoulement de cisaillement

Non-radiative excitation fluorescence microscopy

International audienceNon-radiative Excitation Fluorescence Microscopy (NEFM) constitutes a new way to observe biological samples beyond the diffraction limit. Non-radiative excitation of the samples is achieved by coating the substrate with donor species, such as quantum dots (QDs). Thus the dyes are not excited directly by the laser source, as in common fluorescence microscopy, but through a non-radiative energy transfer. To prevent dewetting of the donor film, we have recently implemented a silanization process to covalently bond the QDs on the substrate. An homogeneous monolayer of QDs was then deposited on only one side of the coverslips. Atomic force microscopy was then used to characterize the QD layer. We highlight the potential of our method through the study of Giant Unilamellar Vesicles (GUVs) labeled with DiD as acceptor, in interaction with surface functionalized with poly-L-lysine. In the presence of GUVs, we observed a quenching of QDs emission, together with an emission of DiD located in the membrane, which clearly indicated that non-radiative energy transfer from QDs to DiD occurs

Nonradiative Excitation Fluorescence Correlation Spectroscopy

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Variable-angle total internal reflection fluorescence microscopy: delving into single cell adhesion

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Non-radiative Excitation Fluorescence Microscopy

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Adhesion induced non-planar and asynchronous flow of a giant vesicle membrane in an external shear flow

International audienceWe show the existence of a flow at the surface of strongly adhering giant lipid vesicles submitted to an external shear flow. The surface flow is divided into two symmetric quadrants and presents two stagnation points (SP) on each side of the vesicle meridian plane. The position of these stagnation points depends strongly on the adhesion strength, characterized by the ratio of the contact zone diameter to the vesicle diameter. Contrary to the case of non-adhesive vesicles, streamlines do not lie in the shear plane. By avoiding the motionless contact zone, streamlines result in three-dimensional paths, strongly asymmetric away from the SP. Additional shearing dissipation may occur on the membrane surface as we observed that the mean rotational velocity of the membrane increases towards the vesicle SP, and is mainly determined by the adhesion induced vesicle shape

Non-radiative Excitation Fluorescence Microscopy for Studying Membrane Adhesion at the Nanoscale

International audienceNon-Radiative Excitation Fluorescence Microscopy (NEFM) constitutes a new way to observe biological samples beyond the diffraction limit. By coating a substrate with an homogeneous monolayer of quantum dots (QDs), Förster Resonance Energy Transfer (FRET) could be achieved between the QDs layer (which play the role of the donor) and Giant Unilamellar Vesicles (GUVs) labelled with DiD (which play the role of the acceptor). The dyes were not directly excited by the laser source but through a non-radiative energy transfer. GUVs were added on a QD layer coated with poly-L-lysine (electrostatic attraction occurred between the positively charged surface and negatively charged GUVs). On this kind of sample, we were able to observe at the same time the emission of the DiD and the quenching of the QDs. It clearly indicates that non-radiative energy transfer occurs from the QDs to DiD. From these two pictures, we also calculated the distance between the lipid membrane and the surface for each pixel with a nanometric resolution