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Figure 2


Fig. 2. Photoactivation of PAGFP-{alpha}Tub84D-expressing embryos. Photoactivation in Drosophila embryos derived from cogGAL4VP16/+;NGT40/+;nanos-GAL4VP16/UASp-PAGFP-{alpha}Tub84D females crossed to UASp-PAGFP-{alpha}Tub84D homozygous males. (A) Photoactivation using Hg 405/20 nm light with a 60x objective. Post-activation fluorescence is approximately proportional to exposure time up to ~30 seconds. (B) Photoactivation using UV confocal laser light (both 351 and 364 nm) and a 63x objective. Fluorescence is approximately proportional to zoom level up to ~16x. (C) Photoactivation is possible with 351 nm UV laser light, stronger with 364 nm light, and strongest with both. (D) UV laser activation of a patch of epidermal cells. PAGFP-Tub is cytoplasmic, excluded from nuclei during interphase, and correctly localises to spindle poles (arrowheads) and mitotic spindle (arrow). (E) A gastrulating embryo after 60 seconds photoactivation using Hg 405/20 nm light. (F,G) z-series reconstruction (F) and z-projection (G) of same embryo imaged using confocal microscopy 15 minutes later, showing that the entire mesoderm is strongly labelled and easily distinguishable from the ectoderm. Scale bars: 10 µm in D; 20 µm in F-G. e, ectoderm; m, mesoderm.