The use of complementary luminescent and fluorescent techniques for imaging Ca ²⁺ signaling events during the early development of zebrafish (danio rerio)

We have visualized many of the Ca ²⁺ signaling events that occur during the early stages of zebrafish development using complementary luminescent and fluorescent imaging techniques. We initially microinject embryos with the luminescent Ca ²⁺ reporter, f-holo-aequorin, and using a custom-designed luminescent imaging system, we can obtain pan-embryonic visual information continually for up to the first ~24 h postfertilization (hpf). Once we know approximately when and where to look for these Ca ²⁺ signaling events within a complex developing embryo, we then repeat the experiment using a fluorescent Ca ²⁺ reporter such as calcium green-1 dextran and use confocal laser scanning microscopy to provide time-lapse series of higher-resolution images. These protocols allow us to identify the specific cell types and even the particular subcellular domain (e.g., nucleus or cytoplasm) generating the Ca ²⁺ signal. Here, we outline the techniques we use to precisely microinject f-holo-aequorin or calcium green-1 dextran into embryos without affecting their viability or development. We also describe how to inject specific regions of early embryos in order to load localized embryonic domains with a particular Ca ²⁺ reporter. These same techniques can also be used to introduce other membrane-impermeable reagents into embryos, including Ca ²⁺ channel antagonists, Ca ²⁺ chelators, fluorescent dyes, RNA, and DNA.