Overestimation of distance traveled by 2D measurements. A) Comparison of MATLAB generated distance maps for 2D and 3D analysis. Increased brightness signifies increased distance from the blood vessel. B) Image slices are thresholded to show highcontrast images of the signals for blood vessel (red), 1st dose (yellow), and 2nd dose (blue). Secondary colors represent overlapping signal. Scale bar is 100 μm and applies to all images. C) Selection of different tumor regions (and different mice) showing how the distance traveled is overestimated when measuring in 2D versus 3D. The box plots represent the spread of 2D distance values for each tumor region. Clearly, there can be multiple 2D distance values (one for each 2D image slice through the volume of each tumor region). The open circles represent the average of all the 2D distance values for each region. The open squares represent the 3D distance values for each region. The dotted and dashed line represent the average 2D and 3D, respectively, distance value for all the different tumor regions. The red rectangle encloses the data points from negative controls. ** p < 0.01.