Robert Pansu

Homo-FRET Microscopy in Living Cells to Measure Monomer-Dimer Transition of GFP-Tagged Proteins

Fluorescence anisotropy decay microscopy was used to determine, in individual living cells, the spatial monomer-dimer distribution of proteins, as exemplified by herpes simplex virus thymidine kinase (TK) fused to green fluorescent protein (GFP). Accordingly, the fluorescence anisotropy dynamics of two fusion proteins (TK27GFP and TK366GFP) was recorded in the confocal mode by ultra-sensitive time-correlated single-photon counting. This provided a measurement of the rotational time of these proteins, which, by comparing with GFP, allowed the determination of their oligomeric state in both the cytoplasm and the nucleus. It also revealed energy homo-transfer within aggregates that TK366GFP progressively formed. Using a symmetric dimer model, structural parameters were estimated; the mutual orientation of the transition dipoles of the two GFP chromophores, calculated from the residual anisotropy, was 44.6 +/- 1.6 degrees, and the upper intermolecular limit between the two fluorescent tags, calculated from the energy transfer rate, was 70 A. Acquisition of the fluorescence steady-state intensity, lifetime, and anisotropy decay in the same cells, at different times after transfection, indicated that TK366GFP was initially in a monomeric state and then formed dimers that grew into aggregates. Picosecond time-resolved fluorescence anisotropy microscopy opens a promising avenue for obtaining structural information on proteins in individual living cells, even when expression levels are very low.

Restrained torsional dynamics of nuclear DNA in living proliferative mammalian cells.

Physical parameters, describing the state of chromatinized DNA in living mammalian cells, were revealed by in situ fluorescence dynamic properties of ethidium in its free and intercalated states. The lifetimes and anisotropy decays of this cationic chromophore were measured within the nuclear domain, by using the ultra-sensitive time-correlated single-photon counting technique, confocal microscopy, and ultra-low probe concentrations. We found that, in living cells: 1) free ethidium molecules equilibrate between extracellular milieu and nucleus, demonstrating that the cation is naturally transported into the nucleus; 2) the intercalation of ethidium into chromatinized DNA is strongly inhibited, with relaxation of the inhibition after mild (digitonin) cell treatment; 3) intercalation sites are likely to be located in chromatin DNA; and 4) the fluorescence anisotropy relaxation of intercalated molecules is very slow. The combination of fluorescence kinetic and fluorescence anisotropy dynamics indicates that the torsional dynamics of nuclear DNA is highly restrained in living cells.

Hyperspectral characterization of fluorescent organic contaminants on optical payloads

The increase of performance of new optical instruments for science and Earth observation always leads to higher requirements in terms of contamination due to particle sedimentation in cleanrooms and deposition of chemical species in vacuum environment. Specific cleanliness control procedures are implemented in order to mitigate the risks of contamination on optical sensors and sensitive diopters, especially when used for UV applications. Such procedures are commonly carried out in cleanrooms and are described in both European ECSS-Q-ST-70-50C and NASA SN-C-0005D standards. UV light at 365 nm is often used for the inspection of optical sensitive surfaces to localize and to evaluate the amount of fluorescent particles, essentially coming from textile fibers. But other groups of compounds can be observed with a different spectral response and distribution, like adhesives and resins or even organic residues. Therefore, we could take advantage of this spectral information closely linked to specific molecules for partial identification of these materials before further investigation involving wipe on flight model and measurement in a laboratory.

Molecular nanocrystals grown in sol-gel thin films for ultrabright chemical sensor applications

Polyaromatic dyes were used to synthesize molecular nanocrystals in sol-gel thin films for sensor applications. Fluorescence Confocal Microscopy (FCM) and Transmission Electron Microscopy (TEM) experiments showed the advantages of our nanocrystallization process compared with microcrystallization in free solutions. Indeed, we obtained well-dispersed and spherical nanocrystals with a narrow size distribution, exhibiting a good crystallinity. Time-resolved fluorescence spectroscopy allowed us to measure fluorescence lifetimes of nanocrystals in presence of molecular probes. Then, chemical sensoring properties of these molecular nanocrystals were demonstrated.