The second set of inflammation imaging experiments employed a paired-agent fluorescence imaging paradigm (Figure ) that intravenously dosed a mouse with a binary mixture of deepred ICB and near-infrared ICG (100 nmol each) and employed dual-channel imaging to simultaneously track the location and clearance of each dye (i.e., 700 nm fluorescence channel for ICB and 800 nm fluorescence channel for ICG). Representative mouse fluorescence images are provided in Figure and Figure with a graph in Figure showing the rise and fall of dye fluorescence intensity in each mouse paw over time (see also the ex vivo biodistribution data in Figure and Figure ). The paired-agent imaging revealed the same trends as the singleagent imaging and demonstrated that the effective perfusion imaging performance of ICG was not altered by binding of a second dye (ICB) to the blood albumin proteins. In other words, coadministration of ICB did not block albumin binding of ICG, which is not surprising since albumin is a highly abundant blood protein. It is also worth emphasizing that the paired-agent imaging paradigm uses half the number of mice as the single-agent imaging method, but it produces the same conclusions concerning dye localization and biodistribution. The successful use of deep-red ICB to visualize blood perfusion within a paired-agent imaging experiment prompted us to pursue a more challenging but more clinically relevant paired-agent imaging paradigm. That is dual-channel imaging of a tumor and its vasculature using a binary admixture of fluorescent probes with different emission wavelengths and targeting properties. We designed a dual-channel imaging experiment that paired ICB, as a deep-red fluorescent probe for visualizing blood perfusion, with a previously reported near-infrared probe called s775cRGD that targets tumor tissue. As illustrated in Figure , a tumor mouse model was established with subcutaneously implanted HT-29 cells in the right flank of the mouse. To account for the difference in probe pharmacokinetics the two probes were administered at different time points.