Figure 2. Registration of space-time image and removal of ‘static’ features.

(A) A one second long raw space-time image from the vessel in Figure 1. Actual image is 608 × 15063 pixels across. Eye motion (due to respiration etc.) causes vessel lumen position to change, as observed in image, making laminar profile measurement challenging if eye motion is not corrected for. For visualization, especially of the static features, image brightness of A–E has been increased by 20%. Scale bars: horizontal = 100 ms, vertical = 40 µm (B) A sample target image, 40 ms long, which is a zoomed-in version of green box in A. For each line in A, information from a symmetric 40 ms window around it is used as the ‘target’ for eye-motion registration. Scale bars: horizontal = 5 ms, vertical = 40 µm. (C) Standard deviation (SD) of pixel values in time dimension (or ‘motion contrast’), is plotted as a function of spatial coordinate. Scale bar: 40 µm. Green and yellow plots correspond to SD profiles of B and E respectively. To register all time points to the same reference lumen position, 1D normalized cross-correlation between SD profiles of target image and a user-defined reference image is used. Thus, eye displacement along fast-scan direction is quantified for each time-point (each line) in space-time image in A. Measured eye motion trace is overlaid on same spatial scale in A (dark red line). This eye motion trace is compared to blood cell velocity trace (measured from same space-time image) later. (D) Registered version of space-time image in A. (E) A 40 ms long reference image, which is a zoomed-in version of yellow box in A. (F) Background subtracted version of image in D. White arrows in A and B shows ‘static’ features (retinal tissue outside lumen) which move much slower than blood cells do. These static features may interfere with slope measurement of moving blood cells. Therefore, a moving-average window of 10 ms is used to subtract the background, leaving only moving blood cells in the image. (G) Background-subtracted version of E. Near horizontal lines due to vessel side-walls, other retinal tissue and specular reflection from top of the vessel have been suppressed.