Preparation and Live Imaging of Regenerating Hydra Spheroids

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.