FUNCTIONAL ULTRASOUND LOCALIZATION MICROSCOPY AND MRI CO-REGISTRATION FOR NEUROVASCULAR ACTIVITY IN ANESTHETIZED NON-HUMAN PRIMATES

Ultrasound Localization Microscopy (ULM) extends functional ultrasound (fUS) to the microvascular scale by localizing microbubble contrast agents, achieving near-capillary resolution and enabling flux quantification. In rodents, functional ULM has shown that time-resolved microbubble flux encodes neurovascular coupling and supports functional connectivity estimation. Translating functional ULM to non-human primates is a critical step, as primate cerebrovascular topology, flow regulation and coupling dynamics more closely reflect human physiology. Compared with BOLD fMRI, fUS provides higher temporal resolution and microvascular specificity using portable and low-cost hardware.
Multi-slice 2D-ULM data were acquired in anesthetized squirrel monkeys (n=4) using a research ultrasound system (6.25 MHz, 128-element probe, 250 × 250 µm²) during 3-minute bolus injections (200 µL Bracco microbubbles + 200 µL saline), as described by Orset et al. (bioRxiv, 2025). ULM slices were co-registered to T2 MRI via rigid affine alignment and demons refinement in MATLAB, enabling backscattering amplitude to be used for 3D microbubble localization in a common MRI space, from which mean flux could also be rendered as a 3D point cloud. The VALiDATe29 atlas was co-registered to subject MRI through centroid pre-alignment, similarity refinement, and demons nonlinear registration, and the resulting transform applied to atlas labels for region definition. Bolus phases were aligned across slices by peak detection to synchronize flux time series, and functional connectivity was computed using correlations across the gray matter regions.
Altogether, this combined fUS-MRI framework demonstrates the feasibility of ULM-based vascular flux and functional connectivity mapping in anesthetized non-human primates.