Overcome the "Buckets Effect": Integration of AIEgens into Proteins for Fluorescence-Enhanced Two-Photon Imaging

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.