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Aggregation induced emission (AIE)-active bright two-photon fluorescent probes with second near-infrared (NIR-II) light excitability can be used for efficient brain bioimaging studies, wherein the fabrication of water-dispersible nanoparticles by encapsulating the hydrophobic probes with amphiphilic polymer holds the key to ensuring biocompatibility and  in vivo  adaptability. However, barely any study has evaluated the structural requirements that can substantially affect the water-dispersible nanoparticle formation ability of an organic AIE-active dye with amphiphilic polymers. The present study systematically assessed the structural dependency of a well-known acrylonitrile based AIE system/fluorogenic core upon the formation of water-dispersible nanoparticles and elucidated how the structural modifications can impact the  in vivo  two-photon imaging.   Methods:  A total of four acrylonitrile-based aggregation induced emission (AIE)-active two-photon (TP) fluorescent probes (AIETP, AIETP C1, AIETP C2 and AIETP C3) have been judiciously designed and synthesized with structural variations to realize how the structural alterations could substantially influence the water-dispersible nanoparticle formation ability (with amphiphilic polymers) and photo-stability to impact the  in vivo  imaging.   Results:  It has been found that the incorporation of the phenyl-thiazole unit in AIETP, AIETP C2 and AIETP C3 facilitated the formation of water-dispersible nanoparticles (NPs) with amphiphilic polymers (Pluronic F127) whereas the presence of only phenyl moiety instead in AIETP C1 could not meet the suitable condition to form the NPs with good aqueous dispersibility. Rationally designed AIETP NPs that exhibited higher brightness, improved photostability and good two-photon absorption cross section was successfully employed for  in vivo  brain vasculature imaging.   Conclusions:  Robust noninvasive 2D and 3D two-photon (NIR-II light, 1040 nm) brain vasculature imaging with beneficial attributes such as outstanding penetration depth (800 µm) and exceptional spatial resolution (1.92 µm), were achieved by utilizing AIETP NPs in this study.

AIE-active two-photon fluorescent nanoprobe with NIR-II light excitability for highly efficient deep brain vasculature imaging