Harnessing Differential Restriction of Molecular Motion within a Single Fluorophore for Versatile Bioimaging

Abstract Numerous studies have highlighted how differently restricted molecular motions within molecular packing can profoundly alter the photophysical properties of fluorophores. However, most investigations remain confined to solid-state luminescence polymorphism, while extending this phenomenon to biological applications remains underexplored. Here, we present a systematic study using N,N-diphenyl-4-(7-phenylbenzo[c][1,2,5]thiadiazol-4-yl) aniline-based derivant, TPABT-P, a turn-on fluorescent probe, to reveal how it can achieve versatile cellular imaging under physiological conditions by different restrictions of molecular motions. Remarkably, beyond its expected prostate-specific membrane antigen (PSMA)-specific binding activation that distinguishes PSMA-expressing cells at both cellular and in vivo levels, TPABT-P can also serve as an excellent cell membrane probe with high contrast and robust photostability. Resolving dual confinement modes can be achieved through adjustments in probe concentration or by employing fluorescence lifetime imaging. By shifting the paradigm of molecular motion confinement from solid states to dynamic living environments, this work provides a powerful insight into deciphering multidimensional fluorescence information, promising a robust toolbox for precise disease diagnostics and real-time biophysical sensing.