Fluorescence imaging is widely used in the life sciences to study biological processes, most often with exogenous fluorescent probes. However, the illumination required to excite these probes also induces photochemical reactions whose mechanisms are often poorly understood. A rigorous understanding of probe photochemistry is essential for their proper use in imaging, for controlling photochemical reactions, and for improving probe design. Since the advent of super-resolution microscopy, probe photochemistry has also been a continuous source of inspiration for the development of innovative imaging methods. Our work on fluorescent probes therefore combines fundamental mechanistic studies with methodological developments in imaging.

Recent work has focused on fluorescent proteins of the GFP family, which are the main probes used for in vivo imaging.

1. Reverse Intersystem Crossing to Reduce Photobleaching and Phototoxicity

Photobleaching and phototoxicity are among the principal limitations of fluorescence imaging. Both processes are generally understood to involve the triplet excited states of fluorescent probes as key intermediates. We recently introduced a method to reduce photobleaching of fluorescent proteins and the associated phototoxicity in vivo under wide-field illumination (Ludvikova et al., Nature Biotechnology, 2024). This approach exploits reverse intersystem crossing (RISC) to depopulate triplet states using near-infrared light. The method can be readily implemented on commercial wide-field microscopes and is effective in both eukaryotic and prokaryotic cells for a broad range of green and yellow fluorescent proteins. Ongoing work aims to extend this strategy to other classes of fluorophores and imaging modalities.

2. Mechanisms of Fluorescent Protein Photoswitching

Reversibly photoswitchable fluorescent proteins (RSFPs) are central probes in super-resolution microscopy and other advanced imaging techniques. X-ray crystallography has revealed two possible photoswitching mechanisms: cis–trans isomerisation of the chromophore coupled to proton transfer, or the reversible addition of a water molecule to the chromophore. We have characterised these two mechanisms in detail using time-resolved spectroscopy down to the femtosecond timescale, focusing on the RSFPs Dreiklang (Renouard et al., J. Phys. Chem. Lett., 2023; Lacombat et al., J. Phys. Chem. Lett., 2017) and Dronpa (Yadav et al., J. Phys. Chem. B, 2015). This work has enabled us to establish a comprehensive mechanistic picture of the elementary steps and characteristic time scales governing fluorescent protein photoswitching.

Experimental Capabilities

We are equipped for mechanistic photochemistry studies with a home-built femtosecond transient absorption spectroscopy setup (excitation 300–700 nm, broadband probe 300–1100 nm, time window 100 fs–3 ns) and a nanosecond optical parametric oscillator (excitation 190–2500 nm) for time-resolved fluorescence and micro- to millisecond transient absorption measurements. These instruments are also routinely used in collaborative projects to investigate molecular systems beyond fluorescent proteins.

Selected References

• Near-infrared co-illumination of fluorescent proteins reduces photobleaching and phototoxicity,
  L. Ludvikova, E. Simon, M. Deygas, T. Panier, M.-A. Plamont, J. Ollion, A. Tebo, M. Piel, L. Jullien, L. Robert, T. Le Saux, A. Espagne,
  Nature Biotechnology, 2024, 42, 872–876.
  [Link]

• Multiscale transient absorption study of the fluorescent protein Dreiklang and two point variants provides insight into photoswitching and non-productive reaction pathways,
  E. Renouard, M. Nowinska, F. Lacombat, P. Plaza, P. Müller, A. Espagne,
  The Journal of Physical Chemistry Letters, 2023, 14, 6477–6485.
  [Link]

• Ultrafast oxidation of a tyrosine by proton-coupled electron transfer promotes light activation of an animal-like cryptochrome,
  F. Lacombat, A. Espagne, N. Dozova, P. Plaza, P. Müller, K. Brettel, S. Franz-Badur, L.-O. Essen,
  Journal of the American Chemical Society, 2019, 141, 13394–13409.
  [Link]

• Photosensitized oxidative addition to gold(I) enables alkynylative cyclization of o-alkylnylphenols with iodoalkynes,
  Z. Xia, V. Corcé, F. Zhao, C. Przybylski, A. Espagne, L. Jullien, T. Le Saux, Y. Gimbert, H. Dossmann, V. Mouriès-Mansuy, C. Ollivier, L. Fensterbank,
  Nature Chemistry, 2019, 11, 797–805.
  [Link]

• Photoinduced chromophore hydration in the fluorescent protein Dreiklang is triggered by ultrafast excited-state proton transfer coupled to a low-frequency vibration,
  F. Lacombat, P. Plaza, M.-A. Plamont, A. Espagne,
  The Journal of Physical Chemistry Letters, 2017, 8, 1489–1495.
  [Link]

• Real-time monitoring of chromophore isomerization and deprotonation during the photoactivation of the fluorescent protein Dronpa,
  D. Yadav, F. Lacombat, N. Dozova, F. Rappaport, P. Plaza, A. Espagne,
  The Journal of Physical Chemistry B, 2015, 119, 2404–2414.
  [Link]