One of the most exciting envisioned applications of PA imaging agents augmented with robust tumor-targeting capabilities is diagnostic cancer imaging. For a dye to be suitable for this purpose it must be minimally cytotoxic. This property was assessed using a standard MTT assay where we found no loss in cell viability up to a dye concentration of 15 µM (6-hour incubation) (Figure ). In addition, after systemic administration, the dye must preferentially localize to the tumor site and the signal enhancement in the lesion should be at least 2-fold. A large dynamic range is important because it will decrease the likelihood of an incorrect assessment. To monitor biodistribution in orthotopic breast tumor-bearing BALB/c mice, we employed fluorescence imaging and found that relative to the initial background scan, the signal enhancement of the tumor site had reached ~2-fold after 8 and 12 hours for mvGlu-AB2 (Figure and) and mvGlu-AB3 (Figure ), respectively. We corroborated these findings by performing PA imaging with spectral unmixing (Figure ). Interestingly, the PA signal appears to span the entire tumor (Figure ), and the difference between the tumor prior to and after injection of mvGlu-AB2 was substantial (4.1-fold) (Figure ). The ability to specifically detect a tumor mass within deep tissue is a testament to the impressive PA properties of our new mvGlu-based dyes. Results obtained for mvGlu-AB3 were comparable and are summarized in the SI (Figures ).