Dynamic Contrast of Reversibly Photoswitchable Fluorescent Labels

Members involved:
, Isabelle AUJARD, Jean-Bernard BAUDIN, Agathe ESPAGNE, Arnaud GAUTIER, Ludovic JULLIEN, Thomas LE SAUX, Marie-Aude PLAMONT,

We recently introduced Out-of-Phase Imaging after Optical Modulation (OPIOM), which exploits reversibly photoswitchable fluorophores as labels and combines optimized periodic illumination with phase-sensitive detection to specifically retrieve the label signal.1-6 OPIOM extracts the fluorescence emission from a targeted label in the presence of autofluorescence, ambient light, or spectrally interfering fluorophores.1,3,4,6 It is also compatible with imaging biological processes in real time.3 OPIOM has been first implemented in fluorescence microscopy.1,3 More recently we demonstrated that OPIOM is relevant as well for other modalities of fluorescence imaging such as macroscale fluorescence imaging4 or wide-field fluorescence microendoscopy.6
OPIOM is powerful but it demands a wide span of photoswitching relaxation times for their discrimination, which is still inconsistent with simultaneous imaging of over 10 labelled biomolecules as anticipated for advanced bioimaging. We currently investigate alternative strategies of dynamic contrast of reversibly photoswitchable fluorophores to reach this goal.
This research is led in collaboration with E. Beaurepaire and W. Supatto (Laboratoire d’Optique et Biosciences, Ecole Polytechnique), V. Croquette (Laboratoire de Physique Statistique, Ecole Normale Supérieure), J.- D. Faure (Institut Jean-Pierre Bourgin, INRA Versailles), A. Lemarchand (Laboratoire de Physique de la Matière Condensée, Sorbonne Université), and J. Livet (Institut de la Vision, Sorbonne Université).

  1. Querard, T.-Z. Markus, M.-A. Plamont, C. Gauron, P. Wang, A. Espagne, M. Volovitch, S. Vriz, V. Croquette, A. Gautier, T. Le Saux, L. Jullien, Photoswitching kinetics and phase sensitive detection add discriminative dimensions for selective fluorescence imaging, Angew. Chem. Int. Ed., 2015, 54, 2633-2637.
  2. Querard, A. Gautier, T. Le Saux, L. Jullien, Expanding discriminative dimensions for analysis and imaging, Chem. Sci., 2015, 6, 2968-2978.
  3. Quérard, R. Zhang, Z. Kelemen, M.-A. Plamont, X. Xie, R. Chouket, I. Roemgens, Y. Korepina, S. Albright, E. Ipendey, M. Volovitch, H. L. Sladitschek, P. Neveu, L. Gissot, A. Gautier, J.-D. Faure, V. Croquette, T. Le Saux, L. Jullien, “Resonant out-of-phase fluorescence microscopy and remote imaging overcome spectral limitations,” Nat. Commun., 2017, 8, 969.
  4. Zhang, R. Chouket, M.-A. Plamont, Zsolt Kelemen, A. Espagne, Alison G. Tebo, A. Gautier, Lionel Gissot, Jean-Denis Faure, L. Jullien, Vincent Croquette, T. Le Saux, “Macroscale fluorescence imaging against autofluorescence under ambient light”, Light Sci. Appl., 2018, 7, 97.
  5. Pellissier-Tanon, R. Chouket, T. Le Saux, L. Jullien, A. Lemarchand, Light-assisted dynamic titration: from theory to an experimental protocol, Phys. Chem. Chem. Phys., 2018, 20, 23998-24010.
  6. Zhang, R. Chouket, Alison G. Tebo, M.-A. Plamont, Z. Kelemen, L. Gissot, J.-D. Faure, A. Gautier, V. Croquette, L. Jullien, T. Le Saux, A simple imaging protocol for autofluorescence elimination and optical sectioning in fluorescence endomicroscopy, Optica, to appear.