Strong Exciton Emission and Ultra‐Photostable Near Infrared‐II Fluorescent Protein for In Vivo Imaging

Abstract In vivo fluorescent imaging in the second near‐infrared window (NIR‐II) provides an excellent approach for understanding the biological processes in substantially scattered tissue environments with reasonable temporal‐spatial resolution. In spite of an enormous amount of organic and inorganic NIR‐II fluorophores developed, there are no reported NIR‐II fluorescent protein. Here, the first NIR‐II fluorescent protein is presented, IRFP1032, which exhibits strong exciton absorption and emission in the NIR‐II region, with exciton extinction coefficient about 4.1 × 10 6 M −1 cm −1 at the excitation maximum 1008 nm, emission maximum of 1032 nm, and emission quantum yield about 0.84%. The IRFP1032 is found to be one of the brightest NIR‐II fluorophores ever reported (brightness of 3.4×10 4 M −1 cm −1 in PBS), thousands‐fold brighter than IR26 in DCM. Furthermore, the IRFP1032 exhibits an ultra‐photostability in comparison to small organic fluorophore. Taking the advantage of the excellent photophysical properties of the NIR‐II fluorescent protein, high‐quality in vivo imaging is realized, for instance, real time observation of blood flow dynamics, dual‐channel imaging of the lymphatic/blood vessel network, and the trajectories of single bacterial cell travelling in blood vessels. The promising NIR‐II in vivo imaging properties demonstrated here with IRPF1032 can open a new scene in fluorescent protein‐based imaging.