Figure 3. Stepwise demonstration of the image analysis method.

The original nuclear Hoechst channel (A) is pre-processed by image sharpening and background subtraction (B), followed by WMC and nuclear mask definition (C). Subsequently, the Voronoi diagram (D) is generated based on the disjointed nuclear masks. For the GFP-p65 channel, the original image (E) is preprocessed by a smoothing filter (F) for global cell location definition (G). By multiplication of the global cell masks (G) with the Voronoi diagram (D), the Voronoi mask is defined for the each cell (H). Within each Voronoi masks the cytoplasmic areas are redefined as the best-fit ellipse in each Voronoi cell (I). Figure (J) shows the composite view of original Hoechst channel, GFP-p65 channel and the BEVC segmentation result.