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

Abstract

<p>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 × 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 one of the brightest NIR-II fluorophores ever reported (brightness of 3.4×104 M−1cm−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.</p>