Third instar larval brains (120 to 144h after egg lying depending on overall growth delay) were dissected in Schneider medium containing 10% FCS and transferred to 50 μL wells (Ibidi, μ-Slide Angiogenesis) for live imaging. Mutant and control brains were imaged in parallel at 25°C. Z-series (thickness of 20 μm with 1 μm spacing) were acquired with a temporal resolution of 30 to 90 s for 1 to 2.5 hours. Alternatively, samples were mounted on a stainless-steel slide, between coverslip and mineral oil as described in a previous study [59 (link)].
Images were acquired with a spinning disk system consisting of a DMi8 microscope (Leica) equipped with a 63X (1.4 N.A.) oil objective, a CSU-X1 spinning disk unit (Yokogawa) and an Evolve EMCCD camera (Photometrics). The microscope was controlled by the Inscoper Imaging Suite and the dedicated software (Inscoper). Alternatively, a CSU-X1 spinning-disk unit mounted on an inverted microscope (Elipse Ti; Nikon) equipped with a 60X (1.4 N.A.) oil objective, a sCMOS ORCA Flash 4.0 (Hamamatsu) and controlled by MetaMorph; was also used for some experiments. Images were processed with Fiji or Imaris softwares.
Images were acquired with a spinning disk system consisting of a DMi8 microscope (Leica) equipped with a 63X (1.4 N.A.) oil objective, a CSU-X1 spinning disk unit (Yokogawa) and an Evolve EMCCD camera (Photometrics). The microscope was controlled by the Inscoper Imaging Suite and the dedicated software (Inscoper). Alternatively, a CSU-X1 spinning-disk unit mounted on an inverted microscope (Elipse Ti; Nikon) equipped with a 60X (1.4 N.A.) oil objective, a sCMOS ORCA Flash 4.0 (Hamamatsu) and controlled by MetaMorph; was also used for some experiments. Images were processed with Fiji or Imaris softwares.
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