In this paper, Urano et al. demonstrate a new class of molecular probe based on a fluorescent small molecule that becomes ‘activated’ after becoming internalized into cells for use in performing in vivo imaging. This probe is developed from an aniline moiety that reacts with protons to produce light-induced electron transfer in a boron-dipyrromethene (BODIPY) fluorophore. Spontaneous relaxation of delocalized electrons to lower energy levels results in fluorescence emission at wavelengths >530 nm, a regime that can be distinguished from autofluorescence background in tissue. This small molecule produces undetectable fluorescence at a physiological pH of 7.4. The probes are ‘switched on’ inside the cell when they encounter an abrupt reduction in pH after being taken up by lysosomes. This novel imaging agent is used with a targeting agent, such as Trastuzumab, an antibody to HER2, a growth factor receptor that is over expressed on the cell surface of many important cancers including breast, esophagus, and lung. Targeting occurs when the Fab component of the probe binds to the HER2 receptor and initiates dimerization. The probe-receptor complex then becomes phosphorylated, internalized by endocytosis, and scavenged by lysosomes. The reduction in pH to a value between 5 and 6 results in the probe releasing an intense fluorescence signal. Furthermore, only live cancer cells are visualized because the low pH in lysosomes requires active proton pumps to maintain an acidic environment. This novel probe is shown to have high specificity for NIH3T3 cells and lung tumors that over express HER2. In addition, activation of the probe after it reaches inside the cell rather than when it is in the extra-cellular space results in a very high target-to-background ratio. This targeted imaging concept can be generalized to the detection of other over expressed cell surface receptors specific to cancer that results in cellular internalization.