Details of segmentation with and without a circularity assumption.Top left: Dapi image. Bottom left:Spot's default segmentation behavior (same color scheme as Fig. 3). Top right: circles placed to maximize the difference in intensity within each circle and outside each circle, all within a box around the estimated spot center. Bottom right: pixels identified by Spot using the circularity assumption exhibit bright red crescents in areas misidentified as containing target material.