Sensitive in vivo imaging of coelenterate luciferases using water-soluble coelenterazine (s-CTZ). A total of 500,000 Gli36-Gluc or Gli36-Rluc cells were injected into the muscle of the left and right leg of nude mice, respectively (n = 4). (a) The mice were injected with 100 μg (4 mg/kg) of native CTZ and imaged using a cooled charge-coupled device camera. Four hours later, when signal from the previous imaging session dropped to background levels, the mice were injected with 100 μg of s-CTZ and imaged again. After another 4 hours, they were imaged after injection of 500 μg of s-CTZ. (b) A typical mouse in which the Gluc-expressing tumor is covered so as to visualize the signal from the Rluc-expressing tumor. (c) Quantitation of tumor-associated signals in a. (d) Blood was collected from the mice, and 5-μl aliquots (in triplicates) were assayed for Gluc activity using 100 μl (50 μg/ml) of either s-CTZ or CTZ and a luminometer. Data are presented as fold differences (in which the signals obtained from native CTZ are set to 1) ± SD (n = 4).