Near Infrared-II Fluorescent protein for In-vivo Imaging

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 is no NIR-II fluorescent protein reported. Here, we present the first NIR-II fluorescent protein, IRFP1032 which exhibits strong exciton absorption and emission in the NIR-II region, with exciton extinction coefficient about 4.1 ×106 M-1cm-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 the brightest NIR-II fluorophore ever reported (brightness of 3.4 × 104 M-1cm-1 in PBS) which is thousands-fold brighter than IR26 in DCM. Taking the advantage of the excellent photo-properties of the NIR-II fluorescent proteins, a collection of high-quality in vivo imaging research was 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. Moreover, a mammalian expression vector was constructed for the IRFP1032, and the corresponding NIR-II fluorescence was able to be recorded unambiguously. The promising NIR-II in vivo imaging properties of IRPF1032 demonstrated here would open a new scene in fluorescent protein-based imaging. ### Competing Interest Statement The authors have declared no competing interest.