All animal studies were approved by the local Institutional Animal Care and Use Committee at Dartmouth College. A series of athymic mice () were each implanted orthotopically with 1 million cells of a U251-green fluorescent protein (GFP) human glioma tumor line. This tumor line was chosen because it is known to overexpress EGFR, enabling specific targeting using an engineered peptide with high affinity for EGFR, the anti-EGFR targeted Affibody (Affibody, Sweden).3,13–15 Transfection of the cell line with GFP allows the GFP signal to be measured ex vivo and provides definitive tumor localization.16 The growth of the tumors was monitored using gadolinium-enhanced T1-weighted magnetic resonance imaging (MRI), which was also the method used to gather anatomical prior information for fluorescence tomography. The general imaging scheme (as shown in Fig. 2) was to record MRI images, perform dual-tracer fluorescence tomographic imaging for up to 1 h, and then euthanize the animal for ex vivo analysis. The animal crania were partially frozen and then cut into thick sections for ex vivo planar fluorescence imaging, allowing spatially resolved quantification of fluorescence probe signal as well as GFP signal from the tumors. The animals were imaged in a staggered fashion, allowing the study of uptake and binding as a function of tumor size as tumor size is generally proportional to time post implantation. Animals were tomographically imaged on days 5, 8, 11, 14, 18, and 21 post tumor implantation. A dual-tracer method was used, with IRDye800CW (LI-COR Biosciences, Lincoln, Nebraska) conjugated to the anti-EGFR Affibody peptide serving as the targeted tracer. Conjugation procedures followed those described by Sexton et al.17 IRDye680RD (LI-COR) conjugated to a nonspecific Affibody peptide (Affibody negative control) was used as the reference tracer. The mice to be imaged were injected via tail vein with 0.1 nmol of each tracer and imaged continuously over the course of , after which time each animal was sacrificed and then imaged ex vivo.