http://www.lkb.ens.fr/recherche/optetbio Optics and Biology Group en-us Glycine is one of the two main inhibitory neurotransmiters. Synaptic plasticity (temporel modification of the synaptic efficiency) depends directly on the number of receptors available in the postsynaptic area. Here we want to understand the role of the membranous diffusion in the temporel regulation of the number of glycine receptors. The use of fluorescent semi-conductor nanocristals allows to follow the movement of individual receptors during several dickers of minutes. Thus it enables us not only to caracterize the diffusif behavior of individual receptors (so that to distinguish different types of population) but also to see changes of comportement within a same trajectory. Thus, we have caraterized their diffusive movement in the neuronal membrane. The fluorescent approach has been completed by electron microscopy imaging of nanocristal-tagged receptors allowing a localization with a nanometrical resolution. Wed, 30 mar 2005 09:00:00 GMT http://www.lkb.ens.fr/recherche/optetbio/Diffusion/page_web_marie_eng.html Stage de M2 : Nous avons une offre de stage pour un etudiant en M2. Le travail porte sur l'etude en molecule unique de mecanismes moleculaires du guidage axonal dans des neurones vivants. Voir details sur le site Wed, 13 oct 2005 12:00:00 GMT http://www.lkb.ens.fr/recherche/optetbio/Stage/index.htm Stage de M2 : Etude par des methodes optiques des interactions entre ADN etire et enzymes de restriction EcoRV a l echelle de la molecule individuelle. Voir details sur le site Wed, 13 oct 2005 12:00:00 GMT http://www.lkb.ens.fr/recherche/optetbio/Stage/index.htm During development of the central nervous system, neurons have to realize accurate connexions so as to constituate a fonctionnal network. Distal part of the axon dedicated to pathfinding is a structure called the growth cone. This allows detection of guidance cues variations, released by other cells of the network. Cytoskeleton (actine and microtubules) is then modified, so as to select the right orientation for axonal growth, until establishment of axon to dendrites connexions. While molecular pathways of this chemotactic activity have been have been widely investigated during the last decade, remarkable robustness of axonal guidance still remains an open question. Feedback from cytoskeleton to guidance cues detection pathway could provide robust guidance and is thus a promissing hypothesis. We are interested in the study of membrane receptors dynamics and its relationship with cytoskeleton. Thanks to semi-conductors nanocrystals - or quantum dots-, we are investigating dynamics of GABAA receptors in membranes of axonal growth cones. Wed, 30 mar 2005 09:00:00 GMT http://www.lkb.ens.fr/recherche/optetbio/ConeCroissance/gc_eng_n.htm In our laboratory we are interested in understanding single molecule dynamics in live cells. For this purpose semiconductor nanocrystals (NCs) represent a suitable visualization tool, since, being more photostable than conventional dyes (like organic fluorophores or fluorescent proteins) they allow imaging over a long period of times. In addition, NCs also have a high sensitivity and resolution, which are critical factors for accurate single molecule tracking. However, since semiconductor nanocrystal application in biological context is still limited by their hydrophobic outer shell we are also interested in understanding their surface chemistry properties. In particular we are focused on the development of new solubilization and conjugate techniques to provide water solubility and binding specificity for these nanoparticles. Wed, 30 mar 2005 09:00:00 GMT http://www.lkb.ens.fr/recherche/optetbio/Solubilisation/index.htm Among the variety of experimental techniques aiming at measuring molecular dynamics in live cells, single particle tracking (SPT) is one of the most sensitive. In this approach, a marker is specifically attached to a protein of interest whose lateral motion is then recorded with high spatial (about 10 nanometers) and temporal resolution (in the millisecond range). Such trajectories contain a wealth of information and they can inform on the local organization of the membrane. In particular, they may potentially reveal transient interactions or temporary confinement that remain hidden with conventional imaging. SPT was initially developped using large labels such as micron-sized latex beads or 40 nm gold nanoparticles. Progress in single molecule biophysics have recently allowed their replacement by smaller fluorescent probes (green fluorescent proteins, fluorophores or quantum dots) that enable investigations at a truly molecular scale. For single molecule tracking (SMT), semiconductor quantum dots (QDs) are probably the most favorable probes since they combine a relatively small size (5-15 nm) with a remarkable brightness and a superior photostability, allowing long-term acquisition with a good signal to noise ratio. While very promising for the study of cell dynamics, the development of SMT raises however new challenges in terms of image processing. In these experiments, the motion of several tagged molecules is simultaneously recorded and needs to be quantitatively analyzed. The image sequences can be composed of up to thousands of frames and contain a number of molecules which varies from a couple to hundreds, depending on experimental conditions and protocols. This number may also fluctuate in consecutives frames since molecules can entry and exit in the field of view. Using QDs as fluorescent probes raises additional difficulties. Due to complex physical processes, the QD fluorescence is intermittent, i.e. its emission intensity randomly alternates between bright and dark periods. It means that tagged-molecules might temporarily disappear from the field of view. A robust processing tool is therefore required to account for all the specificities of SMT measurements and automatically extract the trajectories of single molecules. Wed, 30 mar 2005 09:00:00 GMT http://www.lkb.ens.fr/recherche/optetbio/imageprocessing/index.htm We study by fluorescence microscopy and at a single-molecule level the interactions between combed/stretched DNA and the type II restriction enzymes EcoRI and EcoRV. These enzymes play an important role in the protection of the bacteria against the viruses: they destroy the viral DNA while cutting it in specific sites, thus preventing its insertion in the bacterial DNA and its transcription. Biochemists showed that the restriction enzymes are able to locate their target with a remarkable effectiveness, which cannot be explained by a three-dimensional Brownian diffusion of the enzyme. A mechanism involving a linear diffusion of the enzyme along DNA was thus proposed a few years ago (for a review, see N Shimamoto "One dimensional Diffusion of Proteins along DNA", J Biol. Chem 274, 1990). The direct observation of the dynamics of this process in fluorescence microscopy remains however an experimental challenge on which several teams in the world currently work. To achieve this goal, the molecules of DNA must be placed in a configuration which simultaneously allows their observation and their interaction with proteins. Proteins of interest must also be coupled with fluorophores while preserving their activity. Wed, 30 mar 2005 09:00:00 GMT http://www.lkb.ens.fr/recherche/optetbio/Interaction/index_e.html En cryptographie quantique, la securisation de la transmission de la cle requiert l'utilisation de sources de photons uniques, des sources qui emettent de facon deterministe un photon et un seul, a intervalles de temps reguliers ou sur commande. Pour realiser une telle source, il est necessaire de disposer d'emetteurs uniques faciles a isoler. Notre equipe se propose d'utiliser des nanocristaux semi-conducteurs de CdSe. En cryptographie quantique, la securisation de la transmission de la cle requiert l'utilisation de sources de photons uniques, des sources qui emettent de facon deterministe un photon et un seul, a intervalles de temps reguliers ou sur commande. Pour realiser une telle source, il est necessaire de disposer d'emetteurs uniques faciles a isoler. Notre equipe se propose d'utiliser des nanocristaux semi-conducteurs de CdSe. Leur synthese s'effectue par voie chimique et des parametres tels que la temperature ou le temps permettent de controler leur dimension qui varie entre 1 et 5 nm. Par rapport aux autres dispositifs possibles, ces nanocristaux presentent un grand nombre d'avantages et se placent sans aucun doute parmi les meilleurs candidats pour realiser des sources de photons uniques. Leur stabilite sous irradiation est tres importante (elle peut depasser plusieurs heures). Ils peuvent etre utilises a temperature ambiante. Enfin, les nanocristaux sont faciles a manipuler car disperses en solution. Wed, 30 mar 2005 09:00:00 GMT http://www.spectro.jussieu.fr/Optquant/Semi_conducteurs/Nanocristaux/index_nanocristaux.html