Zebrafish larvae aged 5–9 dpf were embedded in a low-melting-temperature agarose solution at a concentration of 1.8% in embryo medium. In order to minimize movement artifacts, the solution contained 0.3 mg/ml of Pancuronium bromide, a paralyzing agent. The fish was introduced into a glass capillary tube of internal diameter 1.5 mm. The tube was then inserted inside a PMMA square chamber filled with embryo medium and the fish was partially extruded using a piece of plastic tubing inserted in the capillary tube (Figure 1C ). Both sides of the specimen chamber along the illumination path consisted of glass coverslips. The larva dorsoventral axis was aligned vertically by rotation of the agarose cylinder. The chamber was then positioned in the SPIM set-up on a 3-axis manual positioning stage. A piezo-positioner (piezosystem jena PZ 400 OEM) further allowed sub-micrometric vertical displacement of the chamber.
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