Exploiting negative photochromism to harness a four-photon-like fluorescence response with two-photon excitation

Carlos Benitez-Martin, Jean Rouillon, Eduard Fron, Flip de Jong, Morten Grøtli (), Johan Hofkens (), Uwe Pischel () and Joakim Andréasson ()
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Carlos Benitez-Martin: Chalmers University of Technology, Department of Chemistry and Chemical Engineering, Physical Chemistry
Jean Rouillon: Chalmers University of Technology, Department of Chemistry and Chemical Engineering, Physical Chemistry
Eduard Fron: Celestijnenlaan 200F, Chem&Tech – Molecular Imaging and Photonics, KU Leuven
Flip de Jong: Celestijnenlaan 200F, Chem&Tech – Molecular Imaging and Photonics, KU Leuven
Morten Grøtli: University of Gothenburg, Department of Chemistry and Molecular Biology
Johan Hofkens: Celestijnenlaan 200F, Chem&Tech – Molecular Imaging and Photonics, KU Leuven
Uwe Pischel: Campus de El Carmen s/n, CIQSO – Center for Research in Sustainable Chemistry and Department of Chemistry, University of Huelva
Joakim Andréasson: Chalmers University of Technology, Department of Chemistry and Chemical Engineering, Physical Chemistry

Nature Communications, 2025, vol. 16, issue 1, 1-9

Abstract: Abstract Combining nonlinear optical processes and photoswitching transcends the limitations of conventional and even standalone super-resolution imaging. While photoswitching enables resolution improvement, it is typically constrained by limited imaging depth, potential phototoxicity and the low number of inherently fluorescent photoswitches. Nonlinear excitation, such as two-photon absorption, addresses some of these challenges. Here, we present a molecular design strategy that unites the molecular control of T-type negative photoswitches (PS) and two-photon absorption. In these designs, two-photon absorbing push-pull fluorophores that function as FRET-donors are linked to T-type negative PS FRET-acceptors, e.g., donor-acceptor Stenhouse adducts (DASA) or 1,1′-binaphthyl-bridged imidazole dimers. FRET-sensitized isomerization of PS is delicately balanced by reverse thermal isomerization and results in nonlinearly potentiated fluorescence with a quartic fluorescence response upon two-photon excitation, implying enhanced spatial resolution potential. The use of T-type PS is instrumental to this approach, as it ensures temporally stable photonic responses and recyclability without incurring irreversible saturation effects.

Date: 2025
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DOI: 10.1038/s41467-025-66602-1

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