Intravital imaging-based analysis tools for vessel identification and assessment of concurrent dynamic vascular events

Fig. 1. Non-invasive imaging of the ear dermis vasculature. a Mice were anesthetized and immobilized to fix the ear under a water-immersion objective lens for intravital imaging. Cannulation of the tail vein allowed administration of fluorescent labeled dextrans or other carriers into the circulation. A sub-micron glass capillary was used for atraumatic intradermal injection. b Examples of the leakage from blood vessels in response to micro-injection of VEGFA164 but not PBS. Glass capillary was filled with 1 μl VEGFA164 (1 μg/μl) and Alexa633 dye (cyan); about 0.1 μl was injected. Green, 2000 kDa FITC-Dextran. Red, 70 kDa TRITC-Ficoll. Images were acquired 5 min before and 30 min after injection. Bar, 50 μm

Fig. 2. Identification of vessel types in the ear dermis. a Distribution kinetics (frames shown at 0 up to 60 s after injection) of 2000 kDa FITC-Dextran injected as a bolus in the tail vein. Single-photon time-lapse imaging (0.5–1 s/frame) was performed without pinhole to capture a wide focal depth. Bar, 50 μm. b Heatmap of the distribution kinetics of 2000 kDa FITC-Dextran, visualizing arterioles (dark blue), capillaries (green to yellow) and venules (yellow to dark red). c Box plot showing distribution kinetics of 2000 kDa FITC-Dextran for different vessel types, defined as arterioles and venules (>10 μm) and capillaries (≤10 μm), with median (center line), 25th and 75th percentiles (box bounds), and whiskers (maximum and minimum data point) indicated. n = 7 mice. Tukey–Kramer test; ***p < 0.001. d RBC velocity in arterioles, capillaries, and venules in wild-type C57BL/6 ear dermis. n = 7 mice with >20 RBCs measured/vessel-type (10–14 vessels of each type). e Ear dermis vasculature, merged immunostaining for Cldn5 (green), and Isolectin B4 (IB4; red). Boxes labeled i–iii with white dashed vessel outlines are shown as enlarged images below, illustrating consecutive capillary segments…

Fig. 3. RVDM principles and verification. a RBC image is influenced by the speed of laser scanning relative to blood flow velocity and direction resulting in distorted RBC dimensions. b sXYT imaging produces distorted RBC images from which RBC dimensions Xs and Ys and RBC residence time are measured within the scan field (T), thus allowing RBC velocity quantification (see Supplementary Fig. 2 and Methods). c Comparison of RBC velocities estimated from sXYT combined with RVDM, XT, and fXYT image acquisition in the same vessels. Flow angles are indicated for each measurement. n = 3 mice with more than 20 measurements/vessel

Fig. 4. Dynamics of VEGFA-induced vascular leakage. a Point of leakage of circulating 2000 kDa FITC-Dextran and 400 kDa TRITC-Ficoll in a large observation area (left) and zoom-in (right). Flow direction (arrows) and leakage point in a capillary (dashed line), with leakage point boxed (solid line). Right panel, magnification of dashed box area. Capillary leakage point indicated by red arrow. Bar, 200 (left), 100 (right) and 10 μm (inset). b Leakage of 400 kDa Ficoll in response to VEGFA164 in the ear dermis of Cldn5(BAC)-GFP mice. Bar, 100 μm. c Leakage points (red arrow) in Cldn5(BAC)-GFP mouse dermis, in venule (upper) and capillary (lower), showing progressive leakage over time of 400 kDa TRITC-Ficoll in response to VEGFA164. Bar, 10 μm. d Quantification of leakage points/vessel length in the Cldn5(BAC)-GFP ear dermis. Capillaries with GFP expression (positive; pos), mixed (mix) or no GFP (negative; neg) expression were analyzed. Note that Cldn5-expressing capillaries do not leak. N.D.; not detected. n = 3 mice with 6 vessels of each type analyzed/mouse. e Representative leakage point captured by sXYT imaging at −1, 2.5, and 10 min after injection of VEGFA164 (upper and middle…

Fig. 5. Vasodilation and RBC velocity dynamics in response to VEGFA. a Illustration of vasodilation at a leakage point induced in response to VEGFA164 administration. Colors: 2000 kDa FITC-Dextran in lumen; pseudocolor, 2000 kDa FITC-Dextran in dermis; white. White dotted line marks the position used for measurement of vessel diameter dynamics by FWHM at a leakage point (arrow). Bar, 5 μm. b Kinetics of vasodilation at different time points after administration of VEGFA164, in venules (left) and capillaries (right). Gray area around solid black line indicates variability (S.D.) n = 3 mice (individual biological repeats) with 5 vessels of each kind analyzed. Student’s t-test; **p < 0.01. c RBC velocity dynamics in response to VEGFA164 in venules (left) and capillaries (right). Gray area around solid black line indicates variability (S.D.) n = 3 mice and 5 vessels of each kind analyzed with more than 20 RBCs/time point. Student’s t-test; *p < 0.05, **p < 0.01. d Comparison of lag periods for the different responses to VEGFA164, leakage, vasodilation, and velocity changes. n = 20 vessels for leakage analysis in 7 mice and n = 10 vessels in 3 mice (combined capillaries and venules) for…