3D transcranial ultrasound localization microscopy in awake mice: protocol and open-source pipeline

Introduction

Ultrasound localization microscopy (ULM) is the leading technique for super-resolution ultrasound imaging^{1, 2, 3, 4–5} achieving spatial resolutions down to 10 microns. ULM has been widely used to visualize microvascular networks within a variety of organs, including the human brain⁶, heart⁷, and kidneys^{8, 9–10}. ULM is mostly implemented in 2D which represents various limitations such as user dependency, out of plane motion and imprecise quantification¹¹. Volumetric ULM, or 3D ULM^{12, 13–14}, allows appropriate quantification of microbubbles¹⁵ and tissue motion, along with organ-wide acquisitions within the time of a single contrast agent’s bolus (minutes).

To prevent pain, stress, and motion artifacts in animal imaging, animals are routinely scanned with various imaging modalities, including ULM, under anesthesia, such as isoflurane. This anesthetic causes vasodilation and altering flows¹⁶. Natural physiology, stress response, and neuronal plasticity are altered with anesthesia, hindering the translation of functional imaging^17,18. Consequently, the range of relevant biomarkers that could be faithfully extracted from ULM is reduced. The effects of isoflurane-induced anesthesia on cerebral blood flow (CBF) have been extensively studied across various imaging modalities by comparing the anesthetized and awake states. Techniques such as Optical Coherence Tomography have demonstrated significant vasodilation in blood vessels under isoflurane¹⁹, albeit with limitations in depth penetration and field of view due to the inherent properties of optical imaging. Similarly, two-photon microscopy has revealed reductions in CBF through measurements of red blood cell flux²⁰, but this method is also invasive, limited by shallow imaging depth, and requires a craniectomy. On the other hand, Magnetic Resonance (MR) techniques, like Arterial Spin Labeling, provide a non-invasive, volumetric approach to assess CBF²¹, but suffer from poor spatial resolution, particularly at the microvascular level, where precise quantification is critical. Previous work using 2D ULM has demonstrated its potential for awake-state imaging²². This study has relied on initial anesthesia to immobilize the animals for setup, followed by a waiting period for the animal to awaken. However, this approach introduces uncertainty regarding the duration needed for the anesthesia’s effects to fully dissipate, which could significantly impact the accuracy of the awake-state imaging. Additionally, the protocol required invasive procedures such as...