Figure 3. Cell cycle detection and the proliferative landscape of whole lenses. A: EdU-positive (S-phase) cells in the whole lens from an 8-month-old lens. B: Cells undergoing mitosis are labeled using a fluorescent-conjugated antibody to phosphorylated-histoneH3 in an 8-month old lens. C: Total cells in an image field labeled with DRAQ-5. D: Merged image of all three labels detecting two phases of the cell cycle in the germinative zone of a whole lens. E: Whole lenses were placed anterior side down. Image stacks (275 μm) were acquired in the Z-plane from the center of the anterior pole of the lens, demarcated by the asterisk. Z-stacks were then projected in the Y-plane and joined together to re-create the entire lens image. Depth coding was then applied to each stack to provide relevant distance from the initiation point of each z-stack. Cooler colors are closer to the asterisk (origin of the z-stack) than are warmer colors. Using this method, the germinative zone of an entire lens can be seen in a 1-month old lens. F: Reconstruction of an 8-month-old lens showing the decrease in the number of cells in S-phase with age. G: A larger image of the boxed area in (E), showing the color scheme of the depth coding application. H: Larger image of the boxed area in (F). Scale bars: 10 μm (A-D) and 200 μm (E-H).