PA Imaging with mvGlu-AB2. 4T1 cells were trypsinized, neutralized with growth media, and counted on the Countess II FL Cell Counter (Invitrogen, Thermo Fisher Scientific). Cells were spun down, media was aspirated, and a solution was made in 1:1 serum-free RPMI media to Gel-trex™ LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix. Mice were injected in the mammary fat pad with 250,000 cells (50 µL) of the cell solution. Tumors grew for around 1.5-2 weeks and the mice were imaged once tumors reached 100 mm 3 . mvGluAB-2 was reconsituted in 0.9% saline (5% DMSO) and injected retroorbitally. PA images were taken at timepoints = 0 and 6 hours in 5 nm increments from 660-700 nm. Images were spectrally unmixed from hemoglobin, deoxyhemoglobin, and melanin.