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.