Figrue 3 presents representative targeted and untargeted fluorescence images, as well as the corresponding white-light image and a dual-reporter image, in each tumor group at 1 h after intravenous injection of a mixture of EGFR-targeted and untargeted fluorescent reporters (fluorescing at 800 and 700 nm, respectively). In a strictly qualitative sense, for the two tumors that are known to express EGFR (A431 and U251), it was easier to locate the tumor using the dual-reporter image [from Eq. (6)] than with the targeted or untargeted fluorescence images alone at 1 h. Figure 4 elaborates on these observations, presenting a more quantitative analysis. In Fig. 4(a), the average CNRs of targeted fluorescence uptake in A431, U251, 9L-GFP, and blocked U251 tumors are depicted within 1 h after injection of the dual-reporter mixture. A repeated-measures mixed ANOVA demonstrated a significant two-way effect in the data in the form of a significant time-by-tumor line effect (). This suggested that the dynamics of the tumor lines were significantly different; in particular, the U251 tumors tended to exhibit a quicker release of the targeted reporter after injection than the other groups. Despite this effect, however, the between-subject omnibus test suggested that uptake differences were not significant between the tumor lines, at any time point in any tumor line, and no correlation was found between targeted fluorescence uptake and the expected magnitude of EGFR expression in the different tumor lines at any time point. On the other hand, the time courses of the dual-reporter tumor CNRs [depicted in Fig. 3(b)] demonstrated a clear ability to resolve the location of the tumor using dual-reporter imaging at all time points after injection (even at 1 min) for the U251 and A431 tumor lines, and by 20 min after reporter injection for the 9L-GFP tumor line (). The dual-reporter image CNR of the blocked U251 tumors never reached a level of significance over the background. Moreover, by 20 min, the average dual-reporter image value in the tumor [Eq. (6)] measured in each tumor group was significantly different from all other tumor groups, and the difference correlated with the expected differences in EGFR expression between the groups. More specifically, at 20 min, the average tumor dual-reporter CNR in the blocked U251 line, expected to express the least amount of EGFR, was ; in the 9L-GFP line, expected to express a little amount of EGFR, was ; in the U251 line, expected to express a moderate level of EGFR, was ; and in the A431 line, expected to express the most EGFR of the tumor lines, was . To investigate this relationship further, correlation plots were created relating the dual-reporter tumor value at 2 min [Fig. 4(c)] and 1 h [Fig. 4(d)] to the in vivo binding potential, a quantitative marker of receptor expression that has been validated against ex vivo and in vitro measures.21 This approach takes as input the full temporal uptake curve of the targeted and untargeted reporters in the first hour after injection and employs a simplified reference tissue model22 to measure binding potential using the uptake of the untargeted reporter as a “reference tissue.” A statistically significant correlation was observed between the two measures at 20 min and 1 h, with slopes of (, ) and (, ), respectively.