Orca flash 4.0 camera

For live imaging, animals were anesthetized in M9 containing 1mM levamisole and mounted between slide and coverslip on 3% agarose pads. Synchronized L4 animals were treated for 30min in a 96-well flat bottom plate, in 50μl of M9 containing 1mM or 10mM H₂O₂. Treated animals were transferred using a siliconized tip on a freshly seeded plate to recover, and imaged 1h30 to 2h later. Spinning-disk confocal imaging was performed on a system composed of an inverted DMI8 Leica microscope, a Yokogawa CSUW1 head, an Orca Flash 4.0 camera (2048*2018 pixels) piloted by the Metamorph software. Objective used were oil-immersion 40X (HC PL APO, NA 1.3) or 63X (HCX PL APO Lambda blue, NA 1.4). The temperature of the microscopy room was maintained at 20˚C for all experiments. Z-stacks of various body regions were acquired with a constant exposure time and a constant laser power in all experiments. Maximum intensity projections were used to generate the images shown. Fluorescence intensity measurements in int1, I2 and EPC cells were performed using the Fiji software, by manually drawing a region of interest (ROI) around the cell (int1, EPC), or applying a threshold (I2 neurons), background was subtracted and average pixel intensity was quantified.

Images were acquired with an Axio observer Z1 microscope (Zeiss) with a Hamamatsu ORCA-flash 4.0 camera and a spinning disk unit (Yokogawa CSU-X1-A1N-E; Camera evolve, EMCCD 512) with Metamorph software or with a LSM-800 confocal microscope (Zeiss) with Zen software. Images were exported, analyzed and processed with Fiji software. For zx images, xy stacks (0.33 μm z step size) covering cell width were resliced in zx. The quantification of pigmented areas was performed after manual delimitation of culture dish areas using Fiji software. Pictures were then binarized to 8-bit images using a fixed intensity threshold and the black area fraction was measured.