Ultrasound Localization Microscopy for Super-Resolution Mapping of the Rodent Brain Microvasculature

Ultrasound Localization Microscopy (ULM) is a super-resolution imaging technique that enables in vivo visualization of the brain's microvascular architecture surpassing the diffraction limit of conventional ultrasound. By detecting and tracking intravenously injected microbubbles as they circulate through cerebral vessels, ULM produces high-resolution maps of vascular density, flow velocity, and backscattered signal amplitude at spatial scales down to 5-10 µm. This protocol presents a complete workflow for performing ULM imaging in rodents, including animal preparation, probe positioning, image acquisition, microbubble injection, and data processing, using a dedicated functional ultrasound platform. Two preparation methods are described, adapted for mice (transcranial) and rats (with cranial windows), followed by detailed instructions for probe alignment and anatomical targeting using an integrated brain atlas. During acquisition, ultrafast ultrasound sequences are synchronized with bolus injections of microbubbles to capture dynamic flow data. Subsequent reconstruction steps involve clutter filtering, image interpolation, microbubble detection, subpixel localization, and trajectory tracking. Outputs include density maps reflecting vessel occupancy, velocity maps revealing flow patterns and directionality, and amplitude maps offering additional contrast for structural interpretation. Representative results illustrate successful acquisition across full coronal planes and highlight common pitfalls such as poor injection quality, motion artifacts, and skull-induced aberration. The protocol is compatible with both cross-sectional and longitudinal studies and is particularly suited for investigating cerebrovascular alterations in models of aging, stroke, aneurysm, and neurodegenerative diseases. By combining depth penetration, high spatiotemporal resolution, and label-free vascular imaging, ULM offers a powerful tool for noninvasive brain microcirculation analysis in preclinical models.