Indocyanine 7 (Cy7)–tetrameric arginine-glycine-aspartic acid peptide (Cy7 E{E[c(RGDyK)]}) is an integrin-targeted molecular imaging agent developed for near-infrared (NIR) fluorescence imaging of tumor vasculature and tumor angiogenesis (1). Cy7 has a peak excitation wavelength of 743 nm and a peak emission wavelength of 767 nm. Cellular survival, invasion, and migration control embryonic development, angiogenesis, tumor metastasis, and other physiologic processes (2, 3). Among the molecules that regulate angiogenesis are integrins, which comprise a superfamily of cell adhesion proteins that form heterodimeric receptors for extracellular matrix (ECM) molecules (4, 5). These transmembrane glycoproteins consist of two noncovalently associated subunits, α and β (18 α- and 8 β-subunits in mammals), which are assembled into at least 24 α/β pairs. Several integrins, such as integrin αβ, have affinity for the arginine-glycine-aspartic acid (RGD) tripeptide motif, which is found in many ECM proteins. Expression of integrin αβ receptors on endothelial cells is stimulated by angiogenic factors and environments. The integrin αβ receptor is generally not found in normal tissue, but it is strongly expressed in vessels with increased angiogenesis, such as tumor vasculature. It is significantly upregulated in certain types of tumor cells and in almost all tumor vasculature. Molecular imaging probes carrying the RGD motif that binds to the integrin αβ can be used to image tumor vasculature and evaluate angiogenic response to tumor therapy (6, 7). Various RGD peptides in both linear and cyclic forms have been developed for binding to integrin αβ (8). It has been hypothesized that cyclic RGD peptide may have a faster rate of receptor binding or a slower rate of dissociation from the integrin αβ than linear single RGD peptides (9). Wu et al. (9) proposed to evaluated whether a multimeric RGD peptide with >2 repeating cyclic RGD units would further enhance the affinity of the receptor-ligand interactions through a polyvalency effect. They also suggested that the increase in molecular size might prolong circulation time and reduce tumor washout rate. Optical imaging is an imaging method that utilizes light photons emitted from bioluminescence and fluorescence probes (7). Depth penetration is one major limiting factor in optical imaging. Currently, optical imaging has wide applications in small animal imaging but only limited applications in large animal and human studies (10). NIR fluorescence imaging (light range, 650−900 nm) has the advantages of relatively higher tissue penetration and lower autofluorescence from nontarget tissue. NIR fluorescent dyes conjugated RGD peptides such as Cy5.5-c(RGDyK), Cy5.5-c(RGDfK), and Cyp-GRD have been shown to visualize subcutaneously implanted integrin αβ−positive tumors (11-14). Wu et al. (1) investigated the use of the NIR fluorescent dye Cy7−conjugated tetrameric RGD to detect tumor integrin expression. Cy7 was chosen because of its deeper tissue penetration, lower scattering effect, and lower background autofluorescence compared to Cy5.5. The polyvalency effect and suitable apparent size of Cy7-E{E[c(RGDyK)]} appeared to make it a highly potent integrin αβ probe.