Structural and functional mouse brain imaging facilitated by a head-mounted optically transparent skull (HOTS) window allowing offline clearing

High-resolution optical imaging of the cerebral cortex is severely hampered by strong light scattering induced by the opaque skull, posing a major obstacle to deciphering brain structure and function in vivo . Skull optical clearing, a technique that uses chemical cocktails to render the skull transparent, provides direct optical access to the cerebral cortex through the skull. However, existing chemical clearing techniques confer only modest skull transparency due to stringent time constraints and suboptimal chemical recipes. In this work, we developed a head-mounted optically transparent skull (HOTS) window technique. The head-mounted design overcomes clearing time constraints by extending the in vivo skull-clearing process from anesthetized, head-stabilized mice to awake, freely behaving mice. The clearing cocktail was substantially improved over previous recipes via systematic chemical screening tailored to the composition and structure of the skull. Through a series of in vivo and ex vivo experiments, we demonstrated that the HOTS window technique exhibits superior clearing efficacy, good reversibility, favorable biosafety, and broad application prospects. In particular, the HOTS window technique enables transcranial two-photon imaging of neurons at an unprecedented depth, exceeding 800 µm below the pia (permitting visualization layer 5 neurons), in adult mice, substantially outperforming existing skull-clearing methods and the use of thinned-skull windows. The achieved depth even rivals that of open-skull imaging. Even more critically, the HOTS window technique allows sensitive detection of subtle neuronal activity such as calcium transients evoked by whisker stimulation under anesthesia and faint but heterogeneous cAMP dynamics, through the skull, enabling functional investigations that were once restricted to destructive cranial windows.