Figure 5

3D confocal imaging of colon adenoma cultures.

(A) Bright field sectional images at different focal depths (z1 and z2) of colon adenoma cultures showing the different stages of development, namely from small colonospheres, increasing in size to pre-cyst (as the middle starts to collapse) and to a cystic structure. (B–F) Colonospheres and adenoma cysts are shown as RGB 2D images (blue:DAPI, green:β-cat, red:E-cad). (B) 2D overlay images of three small colonospheres increasing in size, showing the low frequency of distinct β-cat-high cells. (C–D) As the colonospheres increase in size, the number of β-cat-high cells increases in frequency. Orthoslice views and 3D reconstruction of two colonospheres (RBG overlays), showing the spherical shape of these structures and the gradual collapse of the top ceiling cell layer when dimensions increases (seen in D). (E) Orthoslice view and sectional 2D views (section 98, 120 and 160) of a large adenoma cyst showing how after collapsing, the structure flattens to form a “bowl” shape. Heterogeneous expressions of β-cat-high cells (green arrows) and E-cad-high cells (red arrows) are clearly identifiable along the surface of the cyst. (inset) A magnified region of the boxed (dotted) region of slice 160 shows clusters of β-cat-high cells and E-cad-high cells. (F) 3D reconstruction of the cyst showing the “bowl-like” shape of the structure. Scale bars: (A–D) 50 μm, (E) 100 μm.