3D Transcranial Ultrasound Localization Microscopy in Awake Mice: Protocol and Open-Source Pipeline

Abstract : Super-resolution ultrasound imaging has emerged as a powerful tool for visualizing microvascular structures and blood flow dynamics beyond the diffraction limit. Despite advances in 3D imaging, most techniques still rely on anesthesia, which significantly alters cerebral hemodynamics. We present an open-source code for 3D super-resolution ultrasound imaging. Open-3DULM was applied to transcranial imaging in habituated awake mice. Comparative analysis reveals that isoflurane anesthesia induces significant vasodilation and increased cerebral blood flow (CBF) in veins, while only a subset of arteries exhibits these effects compared to the awake state. This method could serve as a reference for developing new types of 3D vascular quantifications, particularly emphasizing the importance of awake and 3D imaging for accurate cerebral blood flow measurement in neuroscience research.

Academic reference to be cited : Chabouh, Denis, Abioui-Mourgues, Deloges, Battaglia, Chavignon, Heiles, Pradier, Bodard, Vivien, Orset, and Couture. 3D Transcranial Ultrasound Localization Microscopy in Awake Mice: Protocol and Open-Source Pipeline, Nature Communications Engineering, 2025.

Related scripts and software application : https://github.com/Lab-Imag-Bio/Open-3DULM

Corresponding authors :

• georges.chabouh@sorbonne-universite.fr
• olivier.couture@sorbonne-universite.fr