Overcome the “Buckets Effect”: Integration of AIEgens into Proteins for Fluorescence‐Enhanced Two‐Photon Imaging

Abstract Luminogens with aggregation‐induced emission characteristics (AIEgens) are considered good options for two‐photon (2P) probes, owing to their flexibility of design, heavy‐metal‐free composition, and resistance to photobleaching. However, the design principles for large 2P absorption cross‐section (δ) generally require high coplanarity, strong donor–acceptor (D‐A) interactions, and long conjugation, which can severely weaken the brightness of AIEgens at the aggregated state and undermine their potential in 2P fluorescence imaging (2PFI). Exploration of a feasible approach to overcome the “Buckets Effect” of AIEgen‐based 2P probes is thus a fascinating yet challenging task. Herein, an AIEgen, namely (Z)‐2‐(4‐aminophenyl)‐3‐(5‐(4‐(bis(4‐methoxyphenyl)amino)phenyl)thiophen‐2‐yl)acrylonitrile (MTAA) is designed to have a big δ according to the calculation result and a low fluorescence quantum yield (QY) of 2.2% in dimethyl sulfoxide (DMSO). Impressively, upon integrating into bovine serum albumin (BSA), the protein‐sized MTAA@BSA dots exhibit a 25‐fold higher fluorescence QY compared to MTAA molecules, contributing to an imaging depth of 818 µm in the brain vasculature. The retention and clearance of MTAA@BSA dots in the liver and kidney are also studied using 2PFI. Overall, this work provides a facile approach to overcome the “Buckets Effect” of AIEgen to generate highly efficient, reliable, and biocompatible 2P probes.