Development from a single cell to a complex organism involves cell division, morphogenesis, and formation of 3-D structures of tissues. The traditional characterization of fixed samples bears a few limitations in understanding this highly dynamic process. First, it does not allow us to follow the changes of the same cells or molecules across development due to the lack of temporal information. Second, fixation may introduce artifacts to certain structures. Third, statistics from a larger number of samples are necessary if individual variations exist. With the discovery and development of fluorescent proteins and other fluorescent dyes, imaging from live tissue provides a more straightforward way to visualize molecules or cells of interest in their native environment and collect all dynamic information. However, it is often challenging to get images with good spatial resolution from live tissue due to light distortion by biological samples. Different surgeries are required to expose internal organs before imaging. Additionally, specific immobilization procedures are necessary for imaging moving animals. The goal of this collection is to compile multiple protocols for making preparations for live imaging in developmental biology. We hope it will enable more researchers to use live imaging as a tool to understand developmental processes.