A Two-Photon Ratiometric Sensing Platform Based on Solid-State Luminescent Benzocoumarin: Application to Prolonged Bioimaging of Hydrogen Peroxide.

Fluorescent probes provide essential tools for studying biological systems. For the prolonged imaging of cellular analytes, the fast clearance of small-molecular probes and products is a matter of concern in the quantitative analysis. Activatable probes that produce insoluble products inside cells can be used for prolonged imaging, but those with ratiometric imaging capability are rare. We disclose a novel sensing platform that is capable of prolonged imaging, in addition to ratiometric signaling for reliable quantitative analysis. Specifically, 3-(pyridin-4-yl)-8-(pyrrolidin-1-yl)-2H-benzo[g]chromen-2-one and its pyridinium salt as a product/probe couple constitute the ratiometric sensing platform. As the former dye produces highly emissive insoluble nanoaggregates inside cells, a fluorescent probe in the latter form enables prolonged imaging of the target analyte in cells as well as in tissue by two-photon microscopy.