Bright AIEgen–Protein Hybrid Nanocomposite for Deep and High‐Resolution In Vivo Two‐Photon Brain Imaging

Abstract Two‐photon fluorescence imaging allows in vivo study of biological structures and activities in deep tissues, in which bright fluorophores with high photostability and good biocompatibility are highly desirable. Herein, a small‐molecule fluorogen with aggregation‐induced emission (AIEgen) is complexed with fetal bovine serum (FBS) proteins to develop a protein‐sized AIEgen–protein hybrid nanocomposite (TPEPy‐FBS) with bright far‐red/near‐infrared (NIR) emission, excellent photostability, and low phototoxicity for deep and high‐resolution in vivo two‐photon brain vasculature imaging. Upon complexation with FBS, the fluorescence of TPEPy is greatly intensified and a sixfold enhancement is observed with 10% FBS in aqueous media. The yielded TPEPy‐FBS shows good physical stability in aqueous media and the phototoxicity of TPEPy is dramatically inhibited after complexation with FBS. Moreover, TPEPy‐FBS exhibits bright two‐photon fluorescence in far‐red/NIR region and good photostability upon femtosecond laser excitation, which facilitates high performance in vivo imaging. A large imaging depth of 656 µm is obtained in brain vasculature network imaging with a high signal‐to‐background ratio of 234, where a small blood capillary of 1.05 µm can be resolved at an imaging depth of 656 µm. Highlighted is a simple and versatile strategy to develop efficient two‐photon probes for in vivo biological imaging.