Microscopic features of the human retina can be resolved noninvasively using an adaptive optics scanning laser ophthalmoscope (AOSLO). We describe an improved method to track and quantify the speed of moving objects in AOSLO videos, which is necessary for characterizing the hemodynamics of retinal capillaries. During video acquisition, the objects of interest are in constant motion relative to the background tissue (object motion). The background tissue is in constant motion relative to the AOSLO, due to continuous eye motion during video recordings (eye motion). The location at which AOSLO acquires data is also in continuous motion, since the imaging source is swept in a raster scan across the retina (raster scanning). We show that it is important to take into consideration the combination of object motion, eye motion, and raster scanning for accurate quantification of object speeds. The proposed methods performed well on both experimental AOSLO videos as well as synthetic videos generated by a virtual AOSLO. These methods improve the accuracy of methods to investigate hemodynamics using AOSLO imaging
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