Measurement of circadian rhythms using luminescent circadian clock reporter gene fusions
Seeds were sown on to 0.5 MS 0.8 % agar in PVC tubing rings (0.7 mm diameter) in clusters of 2 - 5 seeds for imaging of luciferase. Plants were grown under same condition as described above for 10-12 days before imaging. Twenty-four and 48 hours before imaging seedlings were dosed with 50 µl of 2 mM luciferin, the substrate of luciferase. Plants were imaged using Photek ICCD225 photon counting cameras using IFS32 software. Automated images were captured every hour for 800 s for luciferase imaging, or for 1500 s every 2 hours for imaging of aequorin. In order to analyse images, captured regions were drawn on a pseudo-coloured image around the sites of clusters using IFS32 software, with the bright field image used as a reference. Total photon counts (luminescence) per image per unit time were extracted. Period estimates were obtained using BRASS software.