Description:
<jats:title>Abstract</jats:title><jats:p>In this paper, we report a new strategy for constructing a dye library with large Stokes shifts. By coupling a dark donor with BODIPY acceptors of tunable high quantum yield, a novel dark resonance energy transfer (DRET)‐based library, named <jats:bold>BNM</jats:bold>, has been synthesized. Upon excitation of the dark donor (<jats:bold>BDN</jats:bold>) at 490 nm, the absorbed energy is transferred to the acceptor (<jats:bold>BDM</jats:bold>) with high efficiency, which was tunable in a broad range from 557 nm to 716 nm, with a high quantum yield of up to 0.8. It is noteworthy to mention that the majority of the non‐radiative energy loss of the donor was converted into the acceptor’s fluorescence output with a minimum leak of donor emission. Fluorescence imaging tested in live cells showed that the <jats:bold>BNM</jats:bold> compounds are cell‐permeable and can also be employed for live‐cell imaging. This is a new library which can be excited through a dark donor allowing for strong fluorescence emission in a wide range of wavelengths. Thus, the <jats:bold>BNM</jats:bold> library is well suited for high‐throughput screening or multiplex experiments in biological applications by using a single laser excitation source.</jats:p>