One goal of molecular imaging is to establish a widely applicable technique for specific detection of tumors with
minimal background. Here, we achieve specific <i>in vivo</i> tumor visualization with a newly-designed "activatable" targeted
fluorescence probe. This agent is activated after cellular internalization by sensing the pH change in the lysosome. Novel
acidic pH-activatable probes based on the BODIPY fluorophore were synthesized, and then conjugated to a
cancer-targeting monoclonal antibody, Trastuzumab, or galactosyl serum albumin (GSA). As proof of concept, <i>ex</i> and <i>in
vivo</i> imaging of two different tumor mouse models was performed: HER2-overexpressed lung metastasis tumor with
Trastuzumab-pH probe conjugates and lectin-overexpressed i.p. disseminated tumor with GSA-pH probe conjugates.
These pH-activatable targeted probes were highly specific for tumors with minimal background signal. Because the
acidic pH in lysosomes is maintained by the energy-consuming proton pump, only viable cancer cells were successfully
visualized. Furthermore, this strategy was also applied to fluorescence endoscopy in tumor mouse models, resulting in
specific visualization of tumors as small as submillimeter in size that could hardly detected by naked eyes because of
their poor contrast against normal tissues. The design concept can be widely adapted to cancer-specific
cell-surface-targeting molecules that result in cellular internalization.