Hydra is renowned for its exceptional regenerative capacity. Remarkably, small regenerating fragments of Hydra tissue, called Hydra spheroids, can establish an organizer de novo . Once established, this organizer directs the surrounding tissue to differentiate into the appropriate oral structures. Within days, the spheroid can develop into a small yet fully patterned polyp. The relatively short timescale of this process enables continuous time-lapse imaging of the entire process, offering a unique opportunity to study the spatiotemporal dynamics of regeneration. Here, we present a detailed workflow and show representative results for generating time-lapse datasets of regenerating Hydra spheroids. The protocol includes a detailed procedure for spheroid generation and guidelines for selecting optimal samples for imaging. We discuss mounting strategies suitable for the long-term imaging of live samples and demonstrate imaging approaches using both spinning disk confocal and light-sheet microscopes. The principles described here are broadly applicable to other model systems in which dynamic morphogenetic processes can be studied using long-term imaging.
Weevers et al. (Fri,) studied this question.