Molecular Engineering for Nonlinear Fluorescence: En Route to Three-Photon Absorption via Sequential One-Photon Excitation

Journal article, 2026

Multiphoton excitation (MPE) processes enable three-dimensionally confined fluorescence with reduced background as well as improved image contrast and signal-to-noise ratio. However, MPE finds major technological limitations derived from the need for high light intensities at long excitation wavelengths. To circumvent these challenges, we herein propose a molecular strategy that reproduces multiphoton-like nonlinear responses using only sequential one-photon excitations (1PE). A dyad (2for1), consisting of the acedan fluorophore covalently connected to a spironaphthopyran photoswitch, shows a quadratic dependence of the emission intensity on the excitation intensity, thus emulating a two-photon absorption behavior. The incorporation of a BODIPY photocage to this construct yields a triad (3for1) that results in an even stronger nonlinear fluorescence response. These findings pave the way for sequential 1PE as a practical approach to capitalize on the benefits of nonlinear fluorescence while avoiding the inherent limitations of simultaneous MPE.