Fig. 3

Real‐time imaging and direct observation of apoptotic cell clearance by microglia during retinal development. A, Diagram of embryonic zebrafish as positioned for live imaging, indicating the developing eye/retina and region subjected to imaging. At the time of imaging, the developing zebrafish eye consists nearly entirely of the lens and retina. Retinas of mpeg1 :mCherry embryos (to label microglia, magenta) beginning at ~54 hours postfertilization (hpf) were imaged live using confocal microscopy. Acridine orange (AO, green) was used to label apoptotic cells in vivo. Images were acquired every 5 minutes, with z stacks spanning the entire retina, for 8 hours total (ending at ~62 hpf). B, shows selected, flattened z projections and selected time‐frames from a representative imaging session. Time stamps (minute:second) are shown at bottom right of each panel, with time 0:00 at the start of the selected frames. C–C′′′′ are enlarged insets of selected regions in B outlined by white boxes, showing mpeg1 :mCherry and AO merged (top row) and AO alone (bottom row). In B and C, a phagosome is formed (arrow in second panel of B, arrow in C) ~25 minutes prior to AO signal detection inside of the phagosome (arrow in fourth panel of B, arrow in C′ top and bottom). Clearance of the AO signal occurs ~40 minutes later (arrow, final panel of B and arrow in C′′′′). Initial AO signal detection was most frequently observed already associated with mpeg1 :mCherry cells (see Figure 4C)