Molecularly engineered high-performance AIE luminogen for NIR-III excitable deep-brain three-photon fluorescence imaging

Three-photon microscopy (3PM) utilizing near-infrared-III (NIR-III) excitation has emerged as an effective technology for deep-tissue bioimaging. Within this domain, organic small molecules with aggregation-induced emission (AIE) property prove to be promising candidates as high-quality 3PM imaging agents because of their exceptional optical properties and the general merit of biocompatibility that most organic small molecules possess. However, due to the bidirectional effects of the intrinsic structure distortion of AIE luminogens (AIEgens) on the three-photon absorption cross section (σ3) and fluorescence quantum yield (η), two crucial factors that determine 3PM performances, the development of high-powered AIE-active 3PM probes with robust ησ3 remains a formidable challenge. In this work, by synergistically integrating multiple beneficial principles into the molecule design, an innovative AIE-active 3PM probe with a symmetrical quadrupole D–A–D architecture, namely TIT, was successfully constructed. Owing to the harmonized planarity and distortion, as well as strong D–A interaction within TIT, the as-prepared TIT nanoparticles (NPs) exhibited boosted ησ3 under 1665 nm NIR-III excitation. This breakthrough enabled brain vascular imaging at a remarkable depth of 1895 μm after craniotomy, thus positioning TIT among the top three organic 3PM probes in terms of imaging depth. In addition, successful hemodynamic imaging at a depth of 1200 μm within the mouse brain post-craniotomy was also accomplished. These achievements establish TIT NPs as highly promising probes for in vivo deep-brain three-photon imaging.