AkaLumine hydrochloride displays greatly enhanced signals emanating from metastasis in the lung, as demonstrated by 8.1-fold higher with AkaLumine hydrochloride as compared with D-luciferin administration. To evaluate the advantage of AkaLumine hydrochloride over CycLuc1 in detecting targets in deep tissue, bioluminescence images are compared between AkaLumine hydrochloride- and CycLuc1-treated mice 15 min after intravenous injection of LLC/luc cells at the dose of 5 mM, which is the maximum concentration of CycLuc1 due to its low water solubility. AkaLumine hydrochloride exhibits 3.3-fold increase in detection sensitivity of disseminated cancer cells in the lung compared with CycLuc1. The advantage of AkaLumine hydrochloride is further confirmed by imaging the same mice bearing lung metastasis after intraperitoneal injection of 5 mM substrates in order of CycLuc1 and AkaLumine hydrochloride, and in the inverse order at an 8-h interval. AkaLumine hydrochloride displays about fourfold increase in signals from lung metastasis compared with CycLuc1^[1].