Three consecutive frames showing a low contrast leukocyte (circled) moving through a human parafoveal capillary. The spatial contrast is very low, since no contrast agents are used; however, by examining consecutive frames, the motion of individual leukocytes can be detected. Small circular dots are photoreceptors. Frames have been corrected for scanning distortion and eye motion. Scale bar, 100 µm.

Two different images generated from the same AOSLO video. (A) Photoreceptor image generated by calculating the average of all frames. (B) Capillary perfusion image generated by applying motion contrast enhancement.

Generation and analysis of a spatiotemporal plot from the AOSLO video shown in Fig. 1. A vessel centerline is identified on the image of perfused capillaries. Vessel coordinates are converted from (x,y) coordinates to (s) coordinates to reduce data by one spatial dimension. To generate the spatiotemporal plot, intensities along s are plotted for each frame of the division video. A 120-frame segment of the spatiotemporal plot for the selected vessel is shown. Sloped traces, which correspond to motion of fluid parcels, are manually extracted. Purely vertical traces are due to artifacts – one example, due to a blink, is shown (arrow). Two example traces that were extracted are shown. The extracted traces were analyzed to give information about flow direction, frequency, speed, and pulsatility as described in the text. F_T1 and F_T2 are the frames at which traces were extracted; F_B’, F_B”, and F_B”’ are the frames at which pulse blips occurred.

Montage showing parafoveal capillaries generated by applying motion contrast enhancement to 76 overlapping AOSLO videos acquired noninvasively without contrast agent. Arrows denote arterioles. Scale bar, 500 µm.

Comparison of AOSLO with red free fundus photography. (A) AOSLO overlay on fundus photograph. The black box is magnified in panels (B) and (C).

Interpretation of spatiotemporal plots, showing representative traces due to (A) plasma gaps and (B) leukocytes. These traces were isolated from full spatiotemporal plots to illustrate the two categories of traces that were identified for analysis. Plasma gap traces are thin and dense, while leukocyte traces are thick, high contrast, sparse, and unidirectional. 200-frame segments of spatiotemporal plots are shown from capillary segments that were identified as a (C) PGC, (D) LPP, and (E) neither a PGC nor a LPP. Note the areas of apparent bidirectionality in the PGC (arrow in C), and artifacts due to poor stabilization when correcting for eye motion (arrows in D) and a blink (arrow in E). Vertical scale bar, 0.5 mm; Horizontal scale bar, 0.5 seconds.

Identification of leukocyte-preferred paths (LPPs) and plasma gap capillaries (PGCs). The distribution of leukocyte and plasma gap frequencies [#/min] are shown across all analyzed capillary segments. Vertical lines are inserted at breaks in the histograms to define LPPs and PGCs.

Spatial distribution of LPPs (green), PGCs (yellow), and all others capillaries that were selected for analysis (gray). Terminal arterioles (red) and collecting venules (blue) are shown for reference.

Examples of averaged velocity waveforms for leukocytes in a single representative LPP segment (top) and plasma gaps in a single representative PGC (bottom), demonstrating the existence of pulsatility in capillaries with single-file flow. Data from extracted leukocytes and plasma gaps are averaged for five equal segments of the cardiac cycle to generate an averaged waveform.