Single-scan adaptive optics-enabled quantitative optical coherence tomography angiography for absolute three-dimensional retinal blood flow mapping

Accurate measurement of blood flow is critical for understanding metabolic function and disease progression, particularly in the retina, where conditions such as diabetic retinopathy, macular degeneration, and glaucoma are closely linked to impairment in microvascular circulation. However, current imaging techniques, including optical coherence tomography angiography (OCTA) and Doppler OCT, do not generally provide direct and absolute blood flow measurements, particularly at the capillary level. Adoption of adaptive optics (AO) and high-speed swept-source lasers in OCT systems has enabled video-rate volumetric acquisition with the ability to resolve individual red blood cells (RBC). Here, we present a quantitative AO-OCTA approach that enables 3D blood flow mapping by integrating OCTA-based vessel morphology with a 3D Radon transform of RBC streaks to measure cell velocity from the raw spatio-temporal OCT data. We describe a complete post-processing pipeline to determine single-cell velocity, vessel diameter, and flow rate using a single scan. We demonstrate depth-resolved flow rates across retinal vessels with diameters from 5 to 120 µm and velocities ranging from 0.5 to 54 mm/s, capturing the wide range of flow dynamics in the human retina. Our methodology is a powerful tool for quantitative blood flow imaging, establishing a robust method for noninvasive, high-resolution microvascular flow quantification with multiple potential applications in biomedical research and clinical diagnostics.