(A) 2D scan pattern of an adaptive optics scanning light ophthalmoscope (AOSLO) that produces a conventional Cartesian or XY image, shown in B. Two orthogonal scanners raster scan the point spread function (PSF) to produce a video (frame rate = 25 Hz, defined by the slow scan rate). (B) A Cartesian image of a first generation arteriole emerging from the optic disk, imaged with 796∆17 nm direct backscatter. The arteriole (intersected by red arrow) is surrounded by nerve fiber bundles. Typically, 250 frames (10 s) of such a video are recorded (Video 1) and averaged to produce the image shown. The scanning field of view was 4.80° x 3.73°. For visualization only, image brightness has been increased by 20–40% in figures of this paper. No brightness or contrast modifications were done for data analysis. Scale bar: vertical = 20 µm (C) To directly image all blood cells without aliasing, the scan pattern was modified to freeze the slow scan at a preferred location intersecting the vessel, shown by the red arrow in B. The PSF is now scanned repeatedly in a 1D path at 15 kHz, enabling high temporal resolution and direct quantitative imaging of all biologically possible blood cell speeds in any vessel size in the mouse retina. (D) Successive 1D scans are stacked horizontally to produce a space-time or XT image, a small snapshot of which is shown. The white streaks are single blood cells in motion, imaged label–free. The slope of these streaks gives the velocity of the cells along the 1D (fast) scan direction. Correcting for the angle of intersection between the 1D scan and the vessel gives the absolute velocity of the individual blood cells (equation in bottom right is Equation 1 in text). Visual inspection of the slopes shows that there are fast moving blood cells at the center of the blood column, and slower cells at the edge. ‘Stationary’ objects, like the nerve fiber bundles, vessel wall or other retinal tissues, manifest as near horizontal features, with near-zero velocity (removed in post-processing by background subtraction, Figure 2). Scale bars: horizontal = 5 ms, vertical = 20 µm. Note: In Figure 1—figure supplement 1, example of capillary flux imaging is shown, with 1D scan placed orthogonal to the capillary to image individual blood cells and determine exact counts of number of cells passing per unit time.