Embryos expressing the indicated fluorescent markers were dechorionated for 2 min in 50% bleach, transferred to a drop of halocarbon oil 27 (Sigma-Aldrich) on a coverslip, and mounted on an oxygen-permeable membrane (YSI). GFP was excited with a 488 nm OPSL (optically pumped semiconductor laser; 2–3.5%) and mCherry was excited with an OPSL 514 nm laser (3–5%). An HCX PL APO 63×/1.4NA CS2 objective on a TCS SP8 Leica resonant scanning confocal microscope (Leica Microsystems) was used for imaging. 12-bit images of one- or two-color Z-stacks (5–13 planes; optical sections: 1.1–1.3 μm) were acquired at 0.45–0.5 μm steps in 4, 6, 10, or 15 s intervals and maximally projected for analysis. Pixel dimensions ranged from 144 to 360 nm/pixel.
To image the consequences of the loss of Dynamin function (DynTS), shits1 embryos expressing ubi-DEcad::GFP or endo-crb::GFP were aged until mid-stage 7 (3h15 AEL) or mid-stage 8 (3h30 AEL) and imaged at 22°C (permissive temperature) or 32°C (restrictive temperature) using a stage top incubator (TOKAI HIT) assembled onto the TCS SP8 Leica resonant scanning confocal microscope.
To image the consequences of the loss of Dynamin function (DynTS), shits1 embryos expressing ubi-DEcad::GFP or endo-crb::GFP were aged until mid-stage 7 (3h15 AEL) or mid-stage 8 (3h30 AEL) and imaged at 22°C (permissive temperature) or 32°C (restrictive temperature) using a stage top incubator (TOKAI HIT) assembled onto the TCS SP8 Leica resonant scanning confocal microscope.