Aggregation-induced emission luminogen for <i>in vivo</i> three-photon fluorescence lifetime microscopic imaging

Compared with visible light, near-infrared (NIR) light has deeper penetration in biological tissues. Three-photon fluorescence microscopy (3PFM) can effectively utilize the NIR excitation to obtain high-contrast images in the deep tissue. However, the weak three-photon fluorescence signals may be not well presented in the traditional fluorescence intensity imaging mode. Fluorescence lifetime of certain probes is insensitive to the intensity of the excitation laser. Moreover, fluorescence lifetime imaging microscopy (FLIM) can detect weak signals by utilizing time-correlated single photon counting (TCSPC) technique. Thus, it would be an improved strategy to combine the 3PFM imaging with the FLIM together. Herein, DCDPP-2TPA, a novel aggregation-induced emission luminogen (AIEgen), was adopted as the fluorescent probes. The three-photon absorption cross-section of the AIEgen, which has a deep-red fluorescence emission, was proved to be large. DCDPP-2TPA nanoparticles were synthesized, and the three-photon fluorescence lifetime of which was measured in water. Moreover, in vivo three-photon fluorescence lifetime microscopic imaging of a craniotomy mouse was conducted via a home-made optical system. High contrast cerebrovascular images of different vertical depths were obtained and the maximum depth was about 600 [Formula: see text]m. Even reaching the depth of 600 [Formula: see text]m, tiny capillary vessels as small as 1.9 [Formula: see text]m could still be distinguished. The three-photon fluorescence lifetimes of the capillaries in some representative images were in accord with that of DCDPP-2TPA nanoparticles in water. A vivid 3D reconstruction was further organized to present a wealth of lifetime information. In the future, the combination strategy of 3PFM and FLIM could be further applied in the brain functional imaging.