It is of great significance to study the brain structure and function in deep-tissue for neuroscience research and bio-medical applications because of the urgent demand for precise theranostics. Three-photon fluorescence microscopic (3PFM) bioimaging excited by the light in near-infrared IIb (NIR-IIb, 1,500–1,700 nm) spectral region is one of the most promising imaging techniques with the advantages of high spatial resolution, large imaging depth, and reduced scattering. Herein, a type of NIR-IIb light excitable deep-red emissive semiconducting polymer dots (P-dots) with bright 3PF and large three-photon absorption cross-section (σ₃) at 1,550 nm was prepared. Then the P-dots were functionalized with polystyrene polymer polystyrene graft ethylene oxide functionalized with carboxyl groups (PS-PEG-COOH) and modified with NH₂-poly(ethylene glycol) (PEG) to synthesis photochemically stable and biocompatible P-dots nanoparticles (NPs). Further the P-dots NPs were utilized for in vivo 3PFM bioimaging of cerebral vasculature with and without the brain skull under 1,550 nm femtosecond (fs) laser excitation. In vivo 3PFM bioimaging of the mice cerebral vasculature at various vertical depths was obtained. Moreover, a vivid three-dimensional structure of the mice vascular architecture beneath the skull was reconstructed. At the depth of 350 µm beneath the brain skull, 3.8 µm blood vessels could still be clearly recognized. NIR-IIb excitable P-dots assisted 3PFM bioimaging has great potential in accurate deep tissue bioimaging.

由于对精确治疗学的迫切需求，研究深层组织的大脑结构和功能对于神经科学研究和生物医学应用具有重要意义。三光子荧光显微镜（3PFM）由近红外IIb（NIR-IIb，1,500–1,700 nm）光谱区域的光激发的生物成像是最有前途的成像技术之一，其优点是空间分辨率高，成像深度大，并减少散射。在本文中，一种类型的NIR-IIB光可激发的深红色发光半导体明亮3PF和大的三光子吸收横截面的聚合物点（P-点）的（σ ₃制备了在1550nm下的）。然后将P点用经羧基官能化的聚苯乙烯聚合物聚苯乙烯接枝环氧乙烷（PS-PEG-COOH）功能化并用NH _2-聚乙二醇（PEG）改性以合成光化学稳定且生物相容的P点纳米颗粒（ NPs）。此外，在1550 nm飞秒（fs）激光激发下，P点NPs用于有或没有脑颅的脑血管的体内3PFM生物成像。体内获得了在不同垂直深度的小鼠脑血管的3PFM生物成像。此外，重建了头骨下方小鼠血管结构的生动三维结构。在脑头骨下方350 µm的深度处，仍可以清晰地识别出3.8 µm的血管。NIR-IIb兴奋性P点辅助3PFM生物成像在精确的深层组织生物成像中具有巨大的潜力。