Abstract
Photoswitchable fluorophores offer precise optical control for advanced imaging, yet the design criteria for an efficient photoswitchable fluorescent dye remain poorly understood. Here, we synthesize four new FRET-based dyads combining different photoswitches with fluorescent dyes and compare them with three previously reported dyads. Dithienylethene-based systems exhibit fluorescence modulation, but diazocine- and fulgimide-based dyads show minimal quenching, despite large FRET overlap. To explain these discrepancies, we develop a model showing that fluorophore absorption at the photoswitching wavelength can induce dye-mediated back-isomerization, reducing the population of the quenching-state. This model allows accurate prediction of photostationary state distributions across all molecular photoswitches and photoswitchable fluorescent dyads and allows us to identify key design principles for high-contrast photoswitchable fluorescent dyes.