Long-term live imaging, cell identification and cell tracking in regenerating crustacean legs

High resolution live imaging of regeneration presents unique challenges, due to the nature of the specimens (large mobile animals), the duration of the process (spanning days or weeks), and the fact that cellular resolution must be achieved without damage caused by lengthy exposures to light. Here, we develop a method for live imaging that captures the entire process of leg regeneration, spanning up to 10 days, at cellular resolution, in the crustaceanParhyale hawaiensis. Our method allows (1) mounting and long-term live imaging of regenerating legs under conditions that yield high spatial and temporal resolution but minimise photodamage, (2) fixing andin situstaining of the regenerated legs that were imaged, to identify cell fates, and (3) computer-assisted cell tracking to determine the cell lineages and progenitors of identified cells. The method is optimised to limit light exposure while maximising tracking efficiency. Combined with appropriate cell-type-specific markers, this method may allow the description of cell lineages for every regenerated cell type in the limb.