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

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 twophoton 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.