Figure 1 Measurement of RBC flux and velocity using confocal line scans. A, A confocal reference image showing the three line scan configurations used for measuring flux and diameter (yellow line), and velocity (red and blue lines). Scale bar, 50 µm. B, A line scan image of a large vessel acquired with a perpendicular line scan (yellow line in A). The y-axis represents time, and the x-axis represents distance. Vessel diameter is measured by imaging FITC-labeled plasma (left), and fRBC flux is measured by imaging fRBCs (right). C, A line scan image of a capillary acquired with a perpendicular line scan (yellow line in A). Vessel diameter and total RBC flux is measured by imaging FITC-labeled plasma (left), and fRBC flux is measured by imaging labeled RBCs (right). D, A line scan image of a large vessel acquired with a line scan parallel to the vessel (red line in A). fRBC velocity is measured from the slope of fRBC streaks, where velocity is inversely proportional to the streak angle. E, A line scan image of a large vessel acquired with a line scan oriented diagonal to the vessel (blue line in A). fRBC velocity across the width of the vessel is measured from the slope of the fRBC streaks along the x-axis of the image. Scale bars in E refer to B–E. Vertical calibration: B, 15.5 ms; C, 77.5 ms; D, 18 ms; E, 30.5 ms. Horizontal scale bars: B, 60.5 µm; C, 10.5 µm; D, 38 µm; E, 121.5 µm.