Abstract

Near-infrared (NIR) genetically encoded fluorescent tags are superior for new imaging capabilities ranging from multiplexed imaging to deep-tissue imaging. Here, we describe the development of NirFAP680; NirFAP680 is an NIR fluorogen-activating protein (FAP) that consists of a newly designed NIR fluorogen termed HBMT and an engineered protein tag termed NirFAP. NirFAP680 has an order of magnitude greater cellular brightness and superior photostability compared to currently available NIR FAPs and fluorescent proteins in both single- and two-photon excitation and allows robust imaging of proteins in live cells and in vivo. Owing to its unique spectroscopic property of a >100 nm Stokes shift, NirFAP680 can also serve as a superb receptor for fluorescence or bioluminescence resonance energy transfer, allowing real-time monitoring of protein–protein interactions in live cells and sensitive bioluminescence imaging in vivo, respectively. Therefore, NirFAP680 will likely be useful for advanced biological imaging in live cells and in vivo.