Figures 3 and 4 show the imaging and quantitative assessment of tumor-bearing mice (PC3-PSMA+ and PC3-PSMA−) administered with PSMA-Cys-Db-ICG, PSMA-Cys-Db-PEG4-ICG, PSMA-Cys-Db-PEG8-ICG, PSMA-Cys-Db-IR700, and J591-ICG using the 700 and 800 nm fluorescence channel of the fluorescence camera for IR700 and ICG, respectively. Covalent conjugation of ICG with short PEG linkers successfully reduced the nonspecific uptake in the liver at early time intervals after injection unlike PSMA-Cys-Db labeled with the original ICG-Sulfo-OSu, which had high liver uptake. However, unexpectedly, there was prolonged high background activity especially in the kidneys, liver, and circulation at 1, 3, and 6 h postinjection with PEG linkers. This resulted in low fluorescence ratios (1.0 to 1.7) of PSMA+ to PSMA− cells for PSMA-Cys-Db-ICG, PSMA-Cys-Db-PEG4-ICG, and PSMA-Cys-Db-PEG8-ICG, although there was a slight improvement in target-to-background ratios after introduction of short PEG linkers to ICG. On the other hand, PSMA-Cys-Db-IR700 (always-on probe) mainly accumulated in the kidneys, but there was low uptake in the liver, which was similar to the biodistribution of intact Cys-Db reported previously.15 The fluorescence ratios of PSMA+ to PSMA− tumors were increased on PSMA-Cys-Db-IR700 and improved over time. As a comparison, J591-ICG selectively accumulated in PC3-PSMA+ tumors within 24 h with low background signal, except in the liver and bowel. The fluorescence intensity in PC3-PSMA+ tumors was maintained on day 3, as reported previously12 (data not shown).