Three dimensional tracking of gold nanoparticles using digital holographic microscopy

In this paper we present a digital holographic microscope to track gold colloids in three dimensions. We report observations of 100nm gold particles in motion in water. The expected signal and the chosen method of reconstruction are described. We also discuss about how to implement the numerical calculation to reach real-time 3D tracking

Holographic microscopy for the three-dimensional exploration of light scattering from gold nanomarkers in biological media

The 3D structure of light scattering from dark-field illuminated live 3T3 cells marked with 40 nm gold nanomarkers is explored. For this purpose, we use a high resolution holographic microscope combining the off-axis heterodyne geometry and the phase-shifting acquisition of the digital holograms. Images are obtained using a novel 3D reconstruction method providing longitudinally undistorted 3D images. A comparative study of the 3D reconstructions of the scattered fields allows us to locate the gold markers which yield, contrarily to the cellular structures, well defined bright scattering patterns that are not angularly titled and clearly located along the optical axis. This characterization is an unambiguous signature of the presence of the gold biological nanomarkers, and validates the capability of digital holographic microscopy to discriminate them from background signals in live cells

Heterodyne holographic microscopy of gold particles

We report experimental results on heterodyne holographic microscopy of subwavelength-sized gold particles. The apparatus uses continuous green laser illumination of the metal beads in a total internal reflection configuration for dark-field operation. Detection of the scattered light at the illumination wavelength on a charge-coupled device array detector enables 3D localization of brownian particles in wate

Parallel heterodyne detection of dynamic light scattering spectra from gold nanoparticles diffusing in viscous fluids.

International audienceWe developed a microscope intended to probe, using a parallel heterodyne receiver, the fluctuation spectrum of light quasi-elastically scattered by gold nanoparticles diffusing in viscous fluids. The cutoff frequencies of the recorded spectra scale up linearly with those expected from single scattering formalism in a wide range of dynamic viscosities (1 to 15 times water viscosity at room temperature). Our scheme enables ensemble-averaged optical fluctuations measurements over multispeckle recordings in low light, at temporal frequencies up to 10 kHz, with a 12 Hz framerate array detector

Detection of single DNA molecules by multicolor quantum-dot end-labeling

Observation of DNA–protein interactions by single molecule fluorescence microscopy is usually performed by using fluorescent DNA binding agents. However, such dyes have been shown to induce cleavage of the DNA molecule and perturb its interactions with proteins. A new method for the detection of surface-attached DNA molecules by fluorescence microscopy is introduced in this paper. Biotin- and/or digoxigenin-modified DNA fragments are covalently linked at both extremities of a DNA molecule via sequence-specific hybridization and ligation. After the modified DNA molecules have been stretched on a glass surface, their ends are visualized by multicolor fluorescence microscopy using conjugated quantum dots (QD). We demonstrate that under carefully selected conditions, the position and orientation of individual DNA molecules can be inferred with good efficiency from the QD fluorescence signals alone. This is achieved by selecting QD pairs that have the distance and direction expected for the combed DNA molecules. Direct observation of single DNA molecules in the absence of DNA staining agent opens new possibilities in the fundamental study of DNA–protein interactions. This work also documents new possibilities regarding the use of QD for nucleic acid detection and analysis

The rotation-coupled sliding of EcoRV

It has been proposed that certain type II restriction enzymes (REs), such as EcoRV, track the helical pitch of DNA as they diffuse along DNA, a so-called rotation-coupled sliding. As of yet, there is no direct experimental observation of this phenomenon, but mounting indirect evidence gained from single-molecule imaging of RE–DNA complexes support the hypothesis. We address this issue by conjugating fluorescent labels of varying size (organic dyes, proteins and quantum dots) to EcoRV, and by fusing it to the engineered Rop protein scRM6. Single-molecule imaging of these modified EcoRVs sliding along DNA provides us with their linear diffusion constant (D1), revealing a significant size dependency. To account for the dependence of D1 on the size of the EcoRV label, we have developed four theoretical models describing different types of motion along DNA and find that our experimental results are best described by rotation-coupled sliding of the protein. The similarity of EcoRV to other type II REs and DNA binding proteins suggests that this type of motion could be widely preserved in other biological contexts

Sliding and jumping of single EcoRV restriction enzymes on non-cognate DNA

The restriction endonuclease EcoRV can rapidly locate a short recognition site within long non-cognate DNA using ‘facilitated diffusion’. This process has long been attributed to a sliding mechanism, in which the enzyme first binds to the DNA via nonspecific interaction and then moves along the DNA by 1D diffusion. Recent studies, however, provided evidence that 3D translocations (hopping/jumping) also help EcoRV to locate its target site. Here we report the first direct observation of sliding and jumping of individual EcoRV molecules along nonspecific DNA. Using fluorescence microscopy, we could distinguish between a slow 1D diffusion of the enzyme and a fast translocation mechanism that was demonstrated to stem from 3D jumps. Salt effects on both sliding and jumping were investigated, and we developed numerical simulations to account for both the jump frequency and the jump length distribution. We deduced from our study the 1D diffusion coefficient of EcoRV, and we estimated the number of jumps occurring during an interaction event with nonspecific DNA. Our results substantiate that sliding alternates with hopping/jumping during the facilitated diffusion of EcoRV and, furthermore, set up a framework for the investigation of target site location by other DNA-binding proteins

Removal of pharmaceutical compounds commonly-found in wastewater through a hybrid biological and adsorption process

[EN] Nowadays, alternative options to conventional wastewater treatment should be studied due to rising concerns emerged by the presence of pharmaceuticals compounds (PhCs) in the aquatic environment. In this work, a combined system including biological treatment by activated sludge plus adsorption with activated carbon is proposed to remove three selected drugs (acetaminophen (ACT), caffeine (CAF) and ibuprofen (IBU)) in a concentration of 2 mg L-1 of each one. For it three sequencing batch reactors (SBR) were operated. SBR-B treated a synthetic wastewater (SWW) without target drugs and SBR-PhC and SBR-PhC + AC operated with SWW doped with the three drugs, adding into SBR-PhC + AC 1.5 g L-1 of a mesoporous granular activated carbon. Results showed that the hybrid system SBR-activated carbon produced an effluent free of PhCs, which in addition had higher quality than that achieved in a conventional activated sludge treatment in terms of lower COD, turbidity and SMP concentrations. On the other hand, five possible routes of removal for target drugs during the biological treatment were studied. Hydrolysis, oxidation and volatilization pathways were negligible after 6 h of reaction time. Adsorption mute only was significant for ACT, which was adsorbed completely after 5 h of reaction, while only 1.9% of CAF and 5.6% of IBU were adsorbed. IBU was the least biodegradable compound.This work was supported by Spanish grants AICO/2018/292 of the Generalitat Valenciana.Ferrer-Polonio, E.; Fernández-Navarro, J.; Iborra-Clar, MI.; Alcaina-Miranda, MI.; Mendoza Roca, JA. (2020). Removal of pharmaceutical compounds commonly-found in wastewater through a hybrid biological and adsorption process. Journal of Environmental Management. 263:1-8. https://doi.org/10.1016/j.jenvman.2020.110368S18263Al-Khazrajy, O. S. A., & Boxall, A. B. A. (2016). Impacts of compound properties and sediment characteristics on the sorption behaviour of pharmaceuticals in aquatic systems. 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Mouvement et dissipation dans une cavité gravitationnelle pour atomes de césium

In the first part of the thesis, we study the motion of atoms in a gravitational cavity. The central element of this cavity is the atomic mirror, formed by an evanescent wave propagating at the surface of a prism of glass. We investigate the expected properties of the mirror and we discuss the losses related to spontaneous emission during the bounce. We then present and analyse our experimental results. In the second part of the thesis, we investigate both theoretically and experimentally an elementary Sisyphus process occuring during the reflection of an atom onto the mirror. This atom may undergo a spontaneous Raman transition between its two hyperfine levels, which leads to an efficient cooling. This cooling allows us to observe very long lifetime in our gravitational cavity. In the third part of the thesis, we investigate theoretically a trap formed by two laser evanescent waves, which confine the atoms in a Morse potential along the direction perpendicular to the prism. We consider a loading process of this trap based on the Sisyphus process previously exposed. We show that it is possible to achieve in this way an efficient loading of the ground state of the Morse potential, and to get thus a quasi bi-dimensional atomic gas at the surface of the dielectric. We then briefly discuss the quantum statistical properties of this gas at very low temperature.La première partie de ce mémoire est consacrée à l'étude d'une cavité gravitationnelle pour atomes de césium. L'élément principal de cette cavité est le miroir à atomes, formé d'une onde évanescente se propageant à la surface d'un prisme de verre. Une étude détaillée des propriétés du miroir et en particulier des phénomènes d'émission spontanée lors du rebond est menée. Les résultats expérimentaux obtenus dans notre groupe sont alors présentés et analysés. La deuxième partie de cette thèse est consacrée à l'étude d'un processus Sisyphe élémentaire susceptible d'intervenir lors du rebond d'un atome sur le miroir. Lors de ce processus, une transition Raman spontanée entre les deux niveaux hyperfins de l'atome entraine un refroidissement particulièrement efficace. Ce refroidissement nous a permis l'observation de longs temps de vie dans la cavité gravitationnelle. Dans la troisième partie de ce mémoire, plus théorique, est proposé un mécanisme de chargement d'un gaz bidimensionnel d'atomes, mécanisme qui repose sur un processus Sisyphe semblable à celui étudié dans la partie précédente. Les atomes du gaz sont piégés au voisinage du prisme sur le niveau fondamental d'un potentiel de Morse réalisé à l'aide de deux ondes évanescentes. Nous montrons que ce mécanisme permet un chargement efficace du piège, et discutons brièvement des propriétés de statistique quantique attendus dans ce gaz bidimensionne