Imaging sequence of calcium waves from 95% epiboly to 12-somite state. Note that bidirectional waves predominate throughout this sequence. The waves in the latter two thirds of the sequence originate near the tailbud region and are referred to here as "tailbud pulses". This sequence covers 5.25 h of development. The movie represents a 40-sec integration window moving in 15-sec steps, running at 150x real time. 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. These 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 for PNAS January 5, 1999 vol. 96: 157-161.