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Hot-band absorption (HBA)-induced anti-Stokes fluorescence (ASF) with longer-wavelength excitation is one effective pathway to deep penetration and low autofluorescence in intravital fluorescence imaging, raising demands for fluorophores with broad spectra, high absorption, and strong emission. However, typical fluorescent dyes display some emission quenching when their concentration is increased in order to obtain brighter fluorescence. In this work, the HBA-induced ASF of aggregation-induced emission (AIE) dots is reported. BPN-BBTD dots were synthesized and confirmed with a fluorescence enhancement and a considerable ASF intensity. In addition, the mechanism of ASF and the HBA process of BPN-BBTD dots were carefully validated and discussed. To obtain the full advantages of the long-wavelength excitation and the short fluorescence lifetime in deep-tissue bioimaging, a large-depth ASF confocal microscopic imaging of in vivo cerebral vasculature was conducted under the excitation of a 980 nm continuous wave laser after intravenous injection of BPN-BBTD dots. Meanwhile, the 3D structure of the cerebrovascular network was successfully reconstructed.

Hot-Band-Absorption-Induced Anti-Stokes Fluorescence of Aggregation-Induced Emission Dots and the Influence on the Nonlinear Optical Effect