Image smoothing and segmentation.

(A) The micrograph shows a cryosection of a spheroid stained with HOECHST (red), Ki67 (blue) and Collagen IV (green). The original image was smoothed with a median filter. For a zoomed-in section of the image (B), the original (C) and smoothed version (D) of the image are visualized as landscapes of the inverted blue color channel intensities, 1 − Iblue. (E) The cell nuclei were segmented from the blue color channel and differentiated between Ki67 positive (red) and negative (blue) nuclei by means of the red channel. The spheroid lumen (green) is approximated by inflated nuclei.