Biomedical subjects

Stéphane Bonneau

Publications and source records attributed to Stéphane Bonneau.

3 recordsLinked to original sources

PubMed2006

Tracking individual proteins in living cells using single quantum dot imaging.

Single quantum dot imaging is a powerful approach to probe the complex dynamics of individual biomolecules in living systems. Due to their remarkable photophysical properties and relatively small size, quantum dots can be used as ultrasensitive detection probes. They make possible the study of biological processes, both in the membrane or in the cytoplasm, at a truly molecular scale and with high spatial and temporal resolutions. This chapter presents methods used for tracking single biomolecules coupled to quantum dots in living cells from labeling procedures to the analysis of the quantum dot motion.

Absorption↗

PubMed2005

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.

Biotinylation↗

PubMed2005

Single quantum dot tracking based on perceptual grouping using minimal paths in a spatiotemporal volume.

Semiconductor quantum dots (QDs) are new fluorescent probes with great promise for ultrasensitive biological imaging. When detected at the single-molecule level, QD-tagged molecules can be observed and tracked in the membrane of live cells over unprecedented durations. The motion of these individual molecules, recorded in sequences of fluorescence images, can reveal aspects of the dynamics of cellular processes that remain hidden in conventional ensemble imaging. Due to QD complex optical properties, such as fluorescence intermittency, the quantitative analysis of these sequences is, however, challenging and requires advanced algorithms. We present here a novel approach, which, instead of a frame by frame analysis, is based on perceptual grouping in a spatiotemporal volume. By applying a detection process based on an image fluorescence model, we first obtain an unstructured set of points. Individual molecular trajectories are then considered as minimal paths in a Riemannian metric derived from the fluorescence image stack. These paths are computed with a variant of the fast marching method and few parameters are required. We demonstrate the ability of our algorithm to track intermittent objects both in sequences of synthetic data and in experimental measurements obtained with individual QD-tagged receptors in the membrane of live neurons. While developed for tracking QDs, this method can, however, be used with any fluorescent probes.

Algorithms↗