Image-based angiography is a well-adapted technique to characterize vasculature, and has been used in retinal neurovascular studies. Because the microvasculature is of particular interest, being the site of exchange between blood and tissue, a high spatio-temporal resolution is required, implying the use of adaptive optics ophthalmoscopes with a high frame rate. Creating the opportunity for decoupled stimulation and imaging of the retina makes the use of near infrared (NIR) imaging light desirable, while the need for a large field of view and a lack of distortion implies the use of a flood illumination-based setup. However, flood-illumination NIR video sequences of erythrocytes, or red blood cells (RBC), have a limited contrast compared to scanning systems and visible light. As a result, they cannot be processed via existing image-based angiography methods. We have therefore developed a new computational method relying on a spatio-temporal filtering of the sequence to isolate blood flow from noise in low-contrast sequences. Applying this computational approach enabled us to perform angiography with an adaptive optics flood illumination ophthalmoscope (AO-FIO) using NIR light, both in bright-field and dark-field modalities. Finally, we demonstrate the capabilities of our system to differentiate blood flow velocity on a retinal capillary network in vivo.
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