Imagem x2 camera

Embryos were imaged on an inverted microscope (UltraVIEW VOX, Perkin Elmer) equipped with a Yokogawa CSU-X1 spinning-disk, an ImagEM-X2 camera (Hamamatsu), 488 nm and 561 nm laser lines for excitation, a Zeiss LD C-Apochromat 40x corrM27 water objective (NA 1.1, WD 0.62 mm at cover glass 0.17 μm) and the Volocity (Perkin Elmer, http://www.perkinelmer.com/) acquisition software. Optical Z planes were spaced by 0.6 μm, the power of the lasers (around 150 mW) and exposure time (between 50 ms and 200 ms) were adjusted depending on the fluorophore and the transgenic line used. In all cases, acquisitions of a Z stacks containing the whole dorsal aorta was reduced to 1 min or less and spaced every 2 min. For TL sequences longer than 1 hr, samples were maintained at 28.5°C using an Okolab cage incubator and the objective was immersed in Immersol W 2010 oil (Zeiss, Cat#: 444969-0000-000).

Unless otherwise indicated, cells were imaged using a Quorum spinning disk microscope with a 10× or 20×, 1.0 NA objectives, or a 63×, 1.4 NA oil immersion objective (Leica DMI6000B inverted fluorescence microscope with a Yokogawa spinning disk head and Hamamatsu ORCA Flash4 sCMOS camera) and Volocity 6.3 acquisition software (Quorum). Confocal z-stacks of 0.3 μm were acquired. Images were analyzed with the Volocity software or Fiji v2.14.0 (ImageJ) and then imported and assembled in Adobe Illustrator v25.3.1 for labeling. For live cell imaging, cells were seeded in μ-Slide 8-well glass bottom chambers (ibidi). Twenty-four hours after seeding, growth media was replaced with live cell imaging media (RPMI with l-Glutamine and 25 mM HEPES (Wisent) supplemented with 10% FBS (Wisent)) containing the respective treatment condition. Cells were imaged at 37 °C using a Leica DMI 6000B inverted fluorescence microscope with a Yokogawa spinning disk head and Hamamatsu ImagEM X2 camera. Images were taken with a z-spacing of 0.5 μm. For invasion ruffle volume measurements, confocal z-stacks of 0.3 μm were acquired.