Visible-near infrared-II skull optical clearing window for in vivo cortical vasculature imaging and targeted manipulation

Skull optical clearing window permits us to perform in vivo cortical imaging without craniotomy, but mainly limits to visible (vis)-near infrared (NIR)-I light imaging. If the skull optical clearing window is available for NIR-II, the imaging depth will be further enhanced. Herein, we developed a vis-NIR-II skull optical clearing agents with deuterium oxide instead of water, which could make the skull transparent in the range of visible to NIR-II. Using a NIR-II excited third harmonic generation microscope, the cortical vasculature of mice could be clearly distinguished even at the depth of 650 μm through the vis-NIR-II skull clearing window. The imaging depth after clearing is close to that without skull, and increases by three times through turbid skull. Furthermore, the new skull optical clearing window promises to realize NIR-II laser-induced targeted injury of cortical single vessel. This work enhances the ability of NIR-II excited nonlinear imaging techniques for accessing to cortical neurovasculature in deep tissue.