A Validation of Spatially Compounded Volumetric Ultrasound Localization Microscopy for Glomerular Imaging With Light Sheet Microscopy

OBJECTIVE: Glomerular structure and microvascular function are central to kidney health, yet current clinical assessments rely largely on indirect serum markers or invasive biopsy. Imaging approaches capable of resolving glomeruli in vivo across the entire kidney remain limited by spatial and temporal resolution. In this study, we perform whole-kidney ultrasound localization microscopy (ULM) with quantification of glomerular microvascular structure and function, and evaluate its performance against light-sheet microscopy (LSM), an established optical gold standard. METHODS: A 5-wk-old rat underwent kidney exposure and stabilization for high-frame-rate ULM imaging. Microbubble contrast agents were continuously infused, and volumetric ultrasound data were acquired at 500 volumes/s using a custom 1024-channel Verasonics system with a 32 × 32 matrix array transducer. ULM processing included beamforming, singular value decomposition filtering, microbubble localization, and tracking to reconstruct renal vasculature. Three spatially registered volumes were combined to form a whole-kidney dataset. Following ultrasound imaging, the kidney was fixed, optically cleared using the iDISCO+ protocol, and imaged with LSM. Non-rigid registration enabled comparison of glomerular density across 100 randomly selected, spatially matched 1 mm³ subvolumes. RESULTS: ULM provided whole-organ imaging with sub-diffraction resolution, enabling visualization of cortical vasculature and individual glomeruli. Glomerular densities measured 21.3 ± 8.0 glomeruli/mm³ with ULM and 28.6 ± 7.5 glomeruli/mm³ with LSM across 100 independent samples, consistent with reported histological values (24.8 ± 0.78 glomeruli/mm³). ULM additionally visualized microvascular flow velocities throughout the kidney. CONCLUSION: This work validates ULM as a proof-of-concept approach for quantitative in vivo assessment of glomeruli, using LSM as the gold standard. These results highlight ULM as a promising approach for whole-kidney, glomerulus-level assessment of renal microstructure and hemodynamics, with potential for future translational applications.