NIR-II Excitable Conjugated Polymer Dots with Bright NIR-I Emission for Deep In Vivo Two-Photon Brain Imaging Through Intact Skull

Methods for noninvasive brain imaging are highly desirable to study brain structures in neuroscience. Two-photon fluorescence microscopy (2PFM) with near-infrared (NIR) light excitation is a relatively noninvasive approach commonly used to study brain with high spatial resolution and large imaging depth. However, most of the current studies require cranial window implantation or skull-thinning methods due to attenuation of excitation light. 2PFM through intact mouse skull is challenging due to strong scattering induced by skull bone. Herein, NIR-II light excitable single-chain conjugated polymer dots (CPdots) with bright fluorescence in NIR-I region (peak at ≈725 nm and quantum yield of 20.6 ± 1.0%) are developed for deep in vivo two-photon fluorescence (2PF) imaging of intact mouse brain. The synthesized CPdots exhibit good biocompatibility, high photostability, and large two-photon absorption cross section. The CPdots allow 2PF images acquired upon excitation at 800, 1040 and 1200 nm with the highest signal-to-background ratio of 208 demonstrated for 1200 nm excitation. Moreover, a 3D reconstruction of the brain blood vessel network is obtained with a large vertical depth of 400 µm through intact skull. This work demonstrates great potential of bright NIR fluorophores for in vivo deep tissue imaging.