Ultrafluorogenic coumarin-tetrazine probes for real-time biological imaging.

We have developed a series of new ultrafluorogenic probes in the blue-green region of the visible-light spectrum that display fluorescence enhancement exceeding 11,000-fold. These fluorogenic dyes integrate a coumarin fluorochrome with the bioorthogonal trans-cyclooctene(TCO)-tetrazine chemistry platform. By exploiting highly efficient through-bond energy transfer (TBET), these probes exhibit the highest brightness enhancements reported for any bioorthogonal fluorogenic dyes. No-wash, fluorogenic imaging of diverse targets including cell-surface receptors in cancer cells, mitochondria, and the actin cytoskeleton is possible within seconds, with minimal background signal and no appreciable nonspecific binding, opening the possibility for in vivo sensing.