Setting up time-lapse experiments on confocal microscopy

GCaMP6f calcium signals and tdTomato signals were captured using a Zeiss LSM 800 confocal microscope. Zeiss Definite Focus 2 was used to avoid focus drift. Time lapse images were acquired using ZEN 2.1 with Time Lapse Module. A single-layer image of 512 × 512 pixels (319.45 µm × 319.45 µm) was acquired every second for 10 min at room temperature (25 °C) with a pixel time of 1.03 µs and fixed laser power, pinhole and other settings for all time-lapse experiments. GCaMP6f emission was recorded at 400-533 nm and tdTomato emission was recorded at 579–700 nm. GCaMP6f and tdTomato fluorescence quantification of each cell was performed manually using ImageJ for each frame. Oscillation frequency was determined by counting individual peaks of the GcaMP6f/tdTomato fluorescence emission ratio observed during 10 min recordings. Heat maps were generated using Matlab. Videos were exported uncompressed from ZEN 2. Genotypes, feeding conditions, scale bars and relative time were added in ZEN 2.