Pre-wave pulse (PWP). The apparently whole-embryo calcium pulse appears to initiate (the PWP region on the wave graphs) over a widespread area of blastoderm, possibly earliest at the animal pole, and then spreads first to the yolk cell membrane and then along the ventral midline heading rostrally. 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. Corresponds to prewave pulse supplemental video in PNAS January 5, 1999 vol. 96: 157-161. See Fig 1 in this paper for quantification of color scale.