Imaging sequence of calcium waves spanning from late shield state to 95% epiboly. This sequence covers 3 h of development and is running at 150x real time.The movie represents a 40-sec integration window moving in 15-sec steps. The playback rate for this and all other movies in the presentation is 10 frames (steps) per second. Fertilized zebrafish eggs were collected within 5 min of spawning, enzymatically dechorionated, and injected with approximately 0.9 nl of a 1% solution of recombinant f-aequorin in 100 mM KCl, 5 mM Mops, and 50 μM EDTA. During imaging the embryos were maintained at 28°C in 30% Danieau's medium containing penicillin (0.5 mg/ml), streptomycin (5,000 units/ml; Sigma), and 0.5% methylcellulose. Imaging was performed on a Photon Imaging Microscope (Science Wares, Falmouth, MA) that used a photon-counting spatial detector with a resistive anode output (Photek, St. Albens-on-Sea, U.K.). Digitized detector output in the form of a stream of time-labeled eight-bit x--y coordinates (256 × 256 pixels) was used to construct time-lapse imaging sequences. The imaging system software allowed the original photon data stream to be analyzed according to any chosen integration time, with the resulting image frames maintaining accurate photon quantitation up to 256 photons per pixel. IThese images are part of an image series within the Zebrafish--The Living Laboratory CD made available by Mark Cooper and described in Methods in Cell Biology Volume 77, 2004, Pages 439-457. This is a supplemental video in PNAS January 5, 1999 vol. 96: 157-161.