Longitudinal Awake Mouse Brain Imaging Using Functional Ultrasound and Functional Ultrasound Localization Microscopy

Ultrafast ultrasound offers a unique route to cross-scale neurovascular phenotyping by integrating functional ultrasound (fUS), ultrasound localization microscopy (ULM), and functional ULM (fULM). Yet the baseline variability, longitudinal stability, and biological safety of such multimodal imaging in awake animals remain insufficiently defined, limiting its use for detecting subtle disease-associated neurovascular changes. Here, an awake longitudinal fUS-ULM-fULM framework is established and validated in mice over five months. Structural vascularity, microvascular flow velocity, mesoscale hemodynamic responses, and microvascular functional responses are repeatedly quantified in the same animals during monthly imaging sessions. Across all metrics, no significant longitudinal drift is detected (p > 0.60). Structural and flow-derived measures are markedly more reproducible than functional readouts, with within-subject coefficients of variation of 5.1% for mean flow velocity and 7.3% for vascularity, compared with 25.0% for fUS-derived cerebral blood volume responses and 53.2% for fULM-derived microvascular functional responses. Mean flow velocity shows the strongest longitudinal consistency (ICC = 0.70) and the lowest detection threshold. Behavioral testing and GFAP/Iba1 staining further reveal no memory impairment or chronic neuroinflammation. This study defines quantitative baselines, reproducibility limits, and safety evidence for awake cross-scale ultrasound imaging, providing a reference framework for longitudinal neurovascular phenotyping in preclinical disease models.