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

Lina Liu; Yufeng Gao; Yuezhi He; Jiuling Liao; Shiwei Ye; Na Xiao; Xueming Cao; Ting Wu; Jia Yu; Wei Zheng; Hui Li

doi:10.3791/71185

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Method Article

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

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DOI:

10.3791/71185

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June 5th, 2026

Lina Liu¹^,² , Yufeng Gao¹^,² , Yuezhi He¹ , Jiuling Liao¹ , Shiwei Ye¹ , Na Xiao¹ , Xueming Cao¹ , Ting Wu¹ , Jia Yu¹ , Wei Zheng¹^,³ , Hui Li¹^,³

¹Research Center for Biomedical Optics and Molecular Imaging, Shenzhen Key Laboratory for Molecular Imaging, Guangdong Provincial Key Laboratory of Biomedical Optical Imaging Technology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, ²University of Chinese Academy of Sciences, ³Key Laboratory of Biomedical Imaging Science and System, Chinese Academy of Sciences, State Key Laboratory of Biomedical Imaging Science and System

Summary

This protocol describes a head-mounted optically transparent skull (HOTS) window based on an optimized two-step skull-clearing procedure. This approach yields a highly transparent skull, enabling two-photon imaging of the adult mouse cerebral cortex to depths of up to ~800 µm.

Abstract

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.

Introduction

Intravital monitoring of cortical neurons is essential for understanding brain structure and function¹. Two-photon microscopy (TPM) is a key tool for such in vivo studies²^,³. However, its imaging quality and depth are severely constrained by strong light scattering from the opaque skull overlying the cortex⁴^,⁵.

Open-skull⁶ and thinned-skull windows⁷^,⁸ have greatly improved optical access to the cortex and are w....