Pierre Desbiolles

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.

Bunching and antibunching in the fluorescence of semiconductor nanocrystals

The fluorescence of single-colloidal CdSe quantum dots is investigated at room temperature by means of the autocorrelation function over a time scale of almost 12 orders of magnitude. Over a short time scale, the autocorrelation function shows complete antibunching, indicating single-photon emission and atomiclike behavior. Over longer time scales (up to tens of seconds), we measure a bunching effect that is due to fluorescence intermittency and that cannot be described by fluctuations between two states with constant rates. The autocorrelation function also exhibits nonstationary behavior related to power-law distributions of On and Off times.

Colloidal CdSe/ZnS quantum dots as single-photon sources

The fluorescence of colloidal CdSe/ZnS nanocrystals at room temperature exhibits a perfect antibunching under continuous or pulsed excitation. In this paper, we discuss the consequences of fluorescence properties of CdSe nanocrystals on the generation of single photons. In particular, we examine the role of Auger processes in the inhibition of multiexcitonic emission. We also discuss the relationship between Auger processes and the fluorescence intermittency of CdSe quantum dots. Altogether, features discussed here indicate that CdSe/ZnS nanocrystals are promising single-photon sources.

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.

Imaging gold nanoparticles in living cell environments using heterodyne digital holographic microscopy

This paper describes an imaging microscopic technique based on heterodyne digital holography where subwavelength-sized gold colloids can be imaged in cell environments. Surface cellular receptors of 3T3 mouse fibroblasts are labeled with 40 nm gold nanoparticles, and the biological specimen is imaged in a total internal reflection configuration with holographic microscopy. Due to a higher scattering efficiency of the gold nanoparticles versus that of cellular structures, accurate localization of a gold marker is obtained within a 3D mapping of the entire samples scattered field, with a lateral precision of 5 nm and 100 nm in the x,y and in the z directions respectively, demonstrating the ability of holographic microscopy to locate nanoparticles in living cell environments.

Dark-field digital holographic microscopy for 3D-tracking of gold nanoparticles

We present a new technique that combines off-axis Digital Holography and Dark Field Microscopy to track 100nm gold particles diffusing in water. We show that a single hologram is sufficient to localize several particles in a thick sample with a localization accuracy independent of the particle position. From our measurements we reconstruct the trajectories of the particles and derive their 3D diffusion coefficient. Our results pave the way for quantitative studies of the motion of single nanoparticle in complex media.

Heterodyne holographic microscopy of gold particles

We report experimental results on heterodyne holographic microscopy of subwavelength-size 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 water.

Tracking of single quantum dot labeled EcoRV sliding along DNA manipulated by double optical tweezers.

Fluorescence microscopy provides a powerful method to directly observe single enzymes moving along a DNA held in an extended conformation. In this work, we present results from single EcoRV enzymes labeled with quantum dots which interact with DNA manipulated by double optical tweezers. The application of quantum dots facilitated accurate enzyme tracking without photobleaching whereas the tweezers allowed us to precisely control the DNA extension. The labeling did not affect the biochemical activity of EcoRV checked by directly observing DNA digestion on the single molecule level. We used this system to demonstrate that during sliding, the enzyme stays in close contact with the DNA. Additionally, slight overstretching of the DNA resulted in a significant decrease of the 1D diffusion constant, which suggests that the deformation changes the energy landscape of the sliding interaction. Together with the simplicity of the setup, these results demonstrate that the combination of optical tweezers with fluorescence tracking is a powerful tool for the study of enzyme translocation along DNA.

Strong evaporative cooling of a trapped cesium gas

Using forced radio-frequency evaporation, we have cooled cesium atoms prepared in the sublevel F = -m(F) = 3 and confined in a magnetic trap. At the end of the evaporation ramp, the sample contains ~ 7000 atoms at 80 nK, corresponding to a phase space density 3 x 10(-2). A molecular dynamics approach, including the effect of gravity, gives a good account for the experimental data, assuming a scattering length larger than 300 Angstrom.

Loading atoms in a bi-dimensional light trap

We investigate theoretically a trap formed by two laser evanescent waves propagating at the surface of a dielectric prism, which confine the atoms in a Morse potential along the direction perpendicular to the prism. We consider a loading process based on the Sisyphus effect, in which a single spontaneous Raman transition is involved. 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.