Circadian Luciferase Imaging in Plants

At dusk of the fourth day of growth, seedlings were sprayed with a 5 mM D-Luciferin (Promega, Madison, WI), 0.01% Triton X-100 solution (Sigma-Aldrich, St. Louis, MO). At dawn of the fifth day, 6–8 seedlings were transferred into a 3- by 3-cm area of the media plate in order to fit inside of the camera’s field of view. Plates were orientated vertically during imaging.
Imaging was performed inside of growth incubators (MIR-154; Panasonic, Japan) at a constant temperature of 22 °C and under an equal mix of red and blue light–emitting diodes (40 μmol m⁻² s⁻¹ total), unless specified as red light only (40 μmol m⁻² s⁻¹ red) or blue light only (40 μmol m⁻² s⁻¹ blue). For experiments under LD cycles, lights were switched on to full intensity at dawn and completely off at dusk. Images were taken every 90 min for 6 d, with an exposure time of 20 min. Images were taken using a LUMO charge-coupled device camera (QImaging, Canada) controlled using Micro-Manager (V2.0; Open Imaging) as previously described [89 (link),90 (link)]. The camera lens (Xenon 25 mm f/0.95; Schneider, Germany) was modified with a 5-mm optical spacer (Cosmicar, Japan) to increase the focal length and decrease the working distance.