English: The various thresholding methods described (Otsu, Huang and Ray) were quantitatively compared to determine the best non-biased method(s) for each imaging type. Two independent reviewers created manual images via cell border identification for each image (I–IV in Figure 1). Images obtained with each thresholding method were then compared to the manually thresholded images, and averaged using various measures found in the literature including: A. the misclassification error, which penalizes misclassified foreground and background pixels in each image; B. total region number nonuniformity, which penalizes images based on incorrect numbers of total regions found; C. region variance nonuniformity, which compares the variance of the segmented region fluorescent intensity between manually thresholded images and the images obtained via the other thresholding methods (Otsu, Huang and Ray); D. The average rank order across six typical intravital images (see Supplemental Fig. 2 for additional images) for each measure (ME, misclassification error; TRNU, region number nonuniformity; VNU, region variance nonuniformity). * p<0.05 relative to Otsu's method, and † p<0.05 relative to Huang's method.