Image-Based Modeling Reveals Dynamic Redistribution of DNA Damage into Nuclear Sub-Domains
Figure 6
Illustration of Rdna and Rgrad Measurements
Three hypothetical foci patterns over the same nucleus are illustrated with their corresponding Rdna and Rgrad values. Upper images (A,C,E) are overlays of the DAPI image with the center of hypothetical foci (in red). Lower images (B,D,F) are overlays of the foci location with the gradient image of DAPI. The gradient operator is often used in imaging as an edge detector. To illustrate this, the green arrow in (C) delineates the contour of the edge of a bright DAPI region. One can see in the corresponding gradient image in (D) that the same contour correlates to a bright gradient region. Rdna measures the ratio of the mean nuclear intensity at the foci locations over the mean intensity of the full nucleus. Rgrad measures the same ratio on the gradient image. Because the boundary of the nuclear image creates a strong gradient intensity, a conservative contour is used for nuclear segmentation (shown in blue) to avoid an edge effect when calculating Rdna and Rgrad. In (A) and (B), foci are placed in areas of surrounding high nuclear density. The surrounding high density keeps the foci distal from areas of density change, thus we see the foci lie in low-intensity regions in the corresponding gradient image. This results in Rdna above 1 and Rgrad below 1. By manually placing foci at different locations with respect to DNA density regions, we show that Rdna is high when foci are located in bright regions of the nucleus (A) and (B); Rgrad is high when foci are located at the interface of bright and dim regions of the nucleus (C) and (D); and Rdna is low when foci are located in dim regions of the nucleus (E) and (F).