A Head-Mounted Optically Transparent Skull (HOTS) Window For Deep Transcranial Imaging of the Mouse Cortex

High-resolution two-photon imaging of the adult mouse cerebral cortex is severely limited by light scattering from the skull, which attenuates signals and restricts imaging depth in vivo. Although skull-clearing methods have been developed to provide optical access to the cortex through the intact skull, their practical performance is constrained by limited clearing time and suboptimal clearing cocktails. Here, we present a detailed protocol for implementing a head-mounted optically transparent skull (HOTS) window. In this approach, a head-mounted cap was used to maintain clearing solutions over the skull, thereby avoiding prolonged anesthesia or physical restraint and enabling extended skull clearing (several hours) in awake, freely behaving mice. Additionally, a two-step clearing procedure was performed using reagents (HOTS-S1: 10% wt/v EDTA, 15% wt/v D-mannose, 10% wt/v sulfolane, 0.5% wt/v Tween 20; HOTS-S2: 70% wt/v D-mannose, 5% wt/v sulfolane, 0.5% wt/v Tween 20) optimized through systematic chemical screening. We provide a step-by-step protocol that includes skull exposure and stabilization, creation and mounting of the head-mounted cap, delivery and refreshment of clearing reagents, and subsequent imaging preparation. In 6-week-old mice (~20 g), the HOTS protocol routinely produces a highly transparent skull that supports two-photon imaging of cortical structures to depths of up to ~800 µm below the pia, approaching the performance of open-skull windows. The HOTS window enables structural imaging in Thy1-GFP-M mice and functional calcium imaging in Thy1-GCaMP6s mice. We believe that, as a convenient and minimally invasive approach, the HOTS window will significantly facilitate deep transcranial imaging and optogenetic, photopharmacological, and other light-based manipulations in vivo.