Generalized photoactivatable fluorescent probes enable background-suppressed 3D single-molecule imaging in hydrogels

Compared with the broad range of regular fluorophores, photoactivatable (PA) fluorophores remain limited in choice, partly due to their commercial availability, complex synthesis routes, and in some case suboptimal photophysical and photochemical properties. Here, we present a type of generalized and modular PA fluorescent probes using DNA as scaffolds. These probes are readily accessible, flexible with fluorophore cores and attachment moieties, and have an improved performance than some existing PA fluorophores. With the probes, we implement 3D single-molecule imaging in hydrogels, overcoming the “concentration barrier” and observing single enzymatic reactions and DNA hybridization events in real time. These versatile PA probes could facilitate a wider adoption of many single-molecule imaging techniques.