Prewave pulse and axial wave. This prewave pulse appears to arise predominantly from the animal pole region, others however appear to emerge nearly simultaneously over large regions of the blastoderm and/or yolk cell membrane. The prewave pulse and the beginning of the wave shown at 20x real time. The movie represents a 30-sec integration window moving in 2-sec steps. 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 ia a supplemental video in PNAS January 5, 1999 vol. 96: 157-161.