Two-photon microscopy is a fluorescence technique particularly well suited for the study of complex molecular biological processes, due to its high sensitivity, excellent temporal resolution, and reduced photodamage in biological environments. In this context, the use of lanthanide based luminescent probes represents a major advantage: their narrow emission bands, spanning from the visible to the infrared region, combined with their long lifetimes, enable time-resolved detection. Furthermore, their conjugation to cell-penetrating peptides induced efficient internalization into cells without inducing significant toxicity. These features are highly beneficial, as they allow the exploitation of the biological transparency window (650–1300 nm) to image cells at depth while overcoming autofluorescence, which exhibits much shorter lifetimes than lanthanide luminescence. The main objective of this thesis was to identify and suppress non-radiative deactivation pathways in a carbazole-based luminescent probe designed for europium sensitization in two-photon microscopy. To this end, a new family of carbazole-based antennas excitable via two-photon absorption was synthesized. This study revealed the impact of introducing electronwithdrawing or electrondonating substituents directly onto the carbazole core, demonstrating that the luminescence of the complex is governed by the competition between photoinduced electron transfer and electronic energy transfer processes responsible for europium sensitization. The introduction of electronwithdrawing substituents yielded highly luminescent probes capable of being internalized by living cells and producing a detectable signal under two-photon microscopy. In addition, optimization of both the probe structure and the cell penetrating peptides enabled efficient internalization in a large proportion of cells. The incorporation of signal peptides further allowed specific targeting of subcellular structures, like the nucleus. Finally, by exploiting both the intrinsic properties of lanthanide complexes and the photoinduced electron transfer process identified in this work, it was possible to design probes sensitive to reactive oxygen species, either through modification of the carbazole substituents or by the introduction of a nitroxide functionality.

Supervision of the thesis:  Olivier SENEQUE (LCBM/PMB) & Jennifer MOLLOY (DCM)