In Vivo 3‐Photon Fluorescence Imaging of Mouse Subcortical Vasculature Labeled by AIEgen Before and After Craniotomy

Abstract Fluorophores lay the material basis for tissue labeling and fluorescence imaging, especially for deep‐brain multiphoton microscopy (MPM) in animal models. Among various fluorescent materials, those with aggregation‐induced emission (AIE) characteristics, i.e., AIEgens, have excellent optical properties and biocompatibility and thus have found widespread applications in biomedical imaging. However, their application to deep‐brain MPM has so far been limited in imaging depth, which undoubtedly poses a hindrance to neurological research aiming to probe the deeper brain. In order to address the issue, here a novel bright AIEgen, namely MTTCM, is designed and synthesized via facile reactions routes. The self‐assembled MTTCM nanoparticles (NPs) with their water‐dispersible feature, have good biocompatibility and good photostability. Furthermore, they are spectrally advantageous for deep‐brain MPM: their emission lies in the NIR‐I region, they generate 3‐photon fluorescence with NIR‐III excitation and show only a slight blue‐shift in the emitted 3‐photon fluorescence in vivo. From a fundamental photochemical perspective, it is also confrimed that MTTCM NPs obey Kasha's rule since the measured 3‐photon and 1‐photon fluorescence spectra overlap. All these merits make MTTCM NPs the enabling fluorophores for record depth in brain imaging in vivo: 1905 µm after craniotomy and 1100 µm through an intact skull, excited at 1660 nm. Furthermore, a record 752 µm hemodynamic imaging depth before craniotomy is demonstrated, from which the blood flow speed can be measured. MTTCM NPs are thus promising fluorophores for deep‐brain 3‐photon imaging in vivo.