Sim scanner

Laser ablation was performed on an Olympus FV1000 confocal microscope equipped with a SIM scanner (Olympus Corporation, Tokyo, Japan). Larvae were immobilized between a slide and a coverslip in NSW containing 100 mM MgCl₂. Larvae were imaged with an UPLSAPO 60X NA:1.20 water immersion objective using a 635-nm laser at 2–5% and transmission imaging with DIC optics. A 351-nm pulsed laser (Teem Photonics™, Grenoble, France) at 8–15% power was used, coupled via air to the SIM scanner for controlled ablation in a region of interest. 12% corresponds to a beam power of 168 µwatts as measured with a microscope slide power sensor (S170C; Thorlabs, Newton, NJ). During eye ablations we also imaged the eye pigment by reflection imaging of the 635-nm light using a PMT. Ablated larvae were placed into NSW for recovery (1–6 hr) before behavioral experiments.

A unit of 2 μg μl⁻¹ of dextran DMNB-caged fluorescein biotin 10,000 molecular weight (synthesized by Invitrogen using funding from NIH R01 grant #DK078209-04S1, Tomoko Obara) was injected into embryos at the one-cell stage. Injected embryos were grown to ∼85% epiboly, dechorionated in 0.5 mg ml⁻¹ pronase from Streptomyces griseus (Roche Diagnostics), washed three times in E3 embryo medium, then individually placed into wells on a Petri dish containing a bed of 1% agarose. Embryos were orientated to a lateral position and fitted to the 306-point grid. Uncaging was performed on the left side of all embryos by selecting a co-ordinate for a 4-s exposure to a 405-nm diode laser using a Sim Scanner on an Olympus FluoView FV1000 confocal laser scanning microscope. As the laser passes through all layers of the embryo, and out the contralateral side, both the epiblast and hypoblast are labelled on either side of the embryo. Uncaged embryos were then imaged and placed in fresh E3 embryo medium for further experimentation. Only the uncaged cells on the left-hand side of the embryo were used for the fate mapping analysis.