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

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 mu m after craniotomy and 1100 mu m through an intact skull, excited at 1660 nm. Furthermore, a record 752 mu m 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.