NONDESTRUCTIVE DETECTION OF FIREFLY LUCIFERASE (LUC) ACTIVITY IN SINGLE PLANT-CELLS USING A COOLED, SLOW-SCAN CCD CAMERA AND AN OPTIMIZED ASSAY

A flexible, comparatively inexpensive system based on a liquid nitrogen-cooled slow-scan CCD (charge coupled device) camera is presented, which can be employed for quantitative low-light (bioluminescence, chemiluminescence or fluorescence) imaging. Using this camera system and the firefly luciferase (LUC) as a screenable marker, transgenic tobacco lines have been produced by direct gene transfer. Bioluminescence emitted from single tobacco cells transiently expressing the firefly luciferase gene (Luc) as well as from stably transformed calli, regenerated shoots, plantlets and T-1 seedlings could be monitored in vivo with no effect on the viability of the material analysed. The patterns of light emission from sections through Luc-expressing leaves and bioluminescent single protoplasts isolated from such leaves were also imaged microscopically. The assay used to detect in vivo LUC activity was optimized by quantifying bioluminescence emitted from Luc-expressing tobacco protoplasts and leaves incubated in different substrate solutions and determining the kinetics of light emission during incubation in the substrate solution.