Currently, a serious problem obstructing the large-scale clinical applications of fluorescence technique is the shallow penetration depth. Two-photon fluorescence microscopic imaging with excitation in the longer-wavelength near-infrared (NIR) region (>1100 nm) and emission in the NIR-I region (650–950 nm) is a good choice to realize deep-tissue and high-resolution imaging. Here, we report ultradeep two-photon fluorescence bioimaging with 1300 nm NIR-II excitation and NIR-I emission (peak ∼810 nm) based on a NIR aggregation-induced emission luminogen (AIEgen). The crab-shaped AIEgen possesses a planar core structure and several twisting phenyl/naphthyl rotators, affording both high fluorescence quantum yield and efficient two-photon activity. The organic AIE dots show high stability, good biocompatibility, and a large two-photon absorption cross section of 1.22 × 103 GM. Under 1300 nm NIR-II excitation, in vivo two-photon fluorescence microscopic imaging helps to reconstruct the 3D vasculature with a high spatial resolution of sub-3.5 μm beyond the white matter (>840 μm) and even to the hippocampus (>960 μm) and visualize small vessels of ∼5 μm as deep as 1065 μm in mouse brain, which is among the largest penetration depths and best spatial resolution of in vivo two-photon imaging. Rational comparison with the AIE dots manifests that two-photon imaging outperforms the one-photon mode for high-resolution deep imaging. This work will inspire more sight and insight into the development of efficient NIR fluorophores for deep-tissue biomedical imaging.

Aggregation-Induced Emission Luminogen with Near-Infrared-II Excitation and Near-Infrared-I Emission for Ultradeep Intravital Two-Photon Microscopy