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Biophotonics and time-resolved fluorescence studies

The biophotonics platform provides specific devices devoted to the study of time-resolved fluorescence parameters (fluorescence lifetime and rotational correlation time, translational diffusion...) for the characterization of small organic compounds or (bio)macromolecules (protein, DNA...). The first set-up allows the measurements of fluorescence intensity/anisotropy decays by time-correlated single photon counting. The analysis of fluorescence intensity decays by the maximum entropy method (1,2) allows the determination of the fluorescence lifetime(s) characterizing the fluorophore of interest.

Using a polarized light as an excitation source, the analysis of fluorescence anisotropy decays allows the determination of rotational correlation times, especially the long correlation time related to the tumbling motion of molecules in the solution. This allows the determination of hydrodynamic volumes of macromolecules and, for instance, the study of the oligomeric status of proteins (1-3).

The second one is a FCS set-up (fluorescence correlation spectroscopy) which allows the study of the translational diffusion of fluorophores or fluorescently labelled molecules (2). The dual colour fluorescence cross-correlation spectroscopy (FCCS) is particularly suitable for the study of molecular interactions (between two interacting species, each labelled with spectrally distinct fluorophores) or the study of the physical separation of two fluorescently labelled molecules, exemplified in the latter case by the measurement of the DNA unwinding activity of helicases (4) (figure).

A 100 femtosecond pulse infra-red and tunable laser (Mai Tai, Spectra Physics, 690-1020nm, 10W) is coupled to two distinct set-ups (1). When coupled to a pulse picker and a second/third harmonic generator (Spectra Physics), the pulsed laser source is used for time-resolved fluorescence studies (i.e. measurements of fluorescence lifetimes and rotational correlation times) of fluorophores emitting in the visible or UV (e.g. tryptophan residues) regions of the spectrum (2).
This laser is also used in the IR mode for biphotonic excitation in FCS or FCCS experiments. It is then coupled to a Nikon TE2000 inverted microscope, one (FCS) or two (FCCS) avalanche photodiodes (Perkin Elmer, SPCM-AQR-14), and a digital correlator (ALV6000) for the calculation of the autocorrelation function or the cross-correlation functions. This laser is also used for the measurement of two-photon fluorescence and the determination of two-photon absorption cross-sections of chemical compounds (5,6).

1. Henry E., Deprez E. & Brochon JC. Maximum entropy analysis of data simulations and practical aspects of time-resolved fluorescence measurements in the study of molecular interactions. J. Mol. Struct. (2014) 1077, 77-86.

2. Deprez E., Tauc P., Leh H., Mouscadet JF., Auclair C., Hawkins M. E. & Brochon JC. DNA binding induces dissociation of the multimeric form of HIV-1 integrase: a time-resolved fluorescence anisotropy study. Proc. Natl. Acad. Sci. U.S.A (2001) 98, 10090-95.

3. Delelis O., Carayon K., Guiot E., Leh H., Tauc P., Brochon J-C., Mouscadet JF. & Deprez E. Insight into the integrase-DNA recognition mechanism: A specific DNA-binding mode revealed by an enzymatically labeled integrase J. Biol. Chem. (2008) 283, 27838-49.

4. Li N., Henry E., Guiot E., Rigolet P., Brochon J-C., Xi XG. & Deprez E. Multiple E. coli helicase monomers cooperate to unwind long DNA substrates: A fluorescence cross-correlation spectroscopy study. J. Biol. Chem. (2010) 285, 6922-6936.

5. Chennoufi R., Bougherara H., Gagey-Eilstein N., Dumat B., Henry H., Subra F., Bury-Moné S., Mahuteau-Betzer F., Tauc P., Teulade-Fichou MP. & Deprez E. Mitochondria-targeted Triphenylamine Derivatives Activatable by Two-Photon Excitation for Triggering and Imaging Cell Apoptosis. Sci. Rep. (2016) 6:21458.

6. Li Y., Wang H., Tarus B., Romero Perez M., Morellato L., Henry E., Berka V., Tsai A.L., Ramassamy B., Dhimane H., Dessy C., Tauc P., Boucher J-L., Deprez E., Slama-Schwok A. Rational design of a fluorescent NADPH derivative imaging constitutive nitric-oxide synthases upon two-photon excitation. Proc. Natl. Acad. Sci. U.S.A (2012) 109, 12526-31.


Equipments List :

  • Spectra Physics Mai Tai laser (690-1020 nm)
  • Pulse Picker and second/third harmonic generator (Spectra Physics)
  • Nikon TE2000 inverted microscope
  • ALV6000 correlator