The cocktail of activatable Alexa Fluor680 (Alexa680)-conjugated panitumumab (Pan) and indocyanine green (ICG)-conjugated trastuzumab (Tra) is a mixture of two self-quenched (SQ), activatable monoclonal antibody (mAb) probes (abbreviated as Pan-Alexa680 and Tra-ICG, respectively) that was developed by Sano et al. for multicolor optical imaging of breast cancer (1). mAbs are widely used to develop optical imaging probes because of their high specificity and affinity to target antigens. However, use of intact mAbs as imaging probes has some disadvantages, such as slow clearance from body, which results in high background and slow penetration of solid tumors (2, 3). To overcome this issue, activatable or “smart” fluorescent probes have been engineered to be silent (fluorescence quenching) but emit signal when or after binding with antigens in the tissue of interest (4, 5). Several photochemical mechanisms of fluorescence quenching and activation have been proposed in the literature, such as homo- (SQ) and hetero-Förster resonance energy transfer (FRET), autoquenching, dimer formation, and photon-induced electron transfer (PeT) (2, 6, 7). Self-quenching of the fluorescence occurs when two excited fluorophores of the same molecule are close enough (<10 nm) to enable them to absorb energy from each other. Hetero-FRET refers to the quenching that occurs between two fluorophores from different molecules (a fluorophore and a quencher molecule). Autoquenching has been observed for some compounds, which can spontaneously induce a quenched state when conjugated with proteins. Autoquenching appears to be induced by the interactions between fluorophores and aromatic rings of amino acids. ICG is a dye that can be fully autoquenched when it is covalently conjugated with mAbs the side chain of lysine, even at a low conjugation ratio. Some compounds can form H- or J-homodimers at high concentrations (~mM) in aqueous solutions and quench the emission fluorescence signal. PeT is used for the fluorescence quenching that happens within a single fluorophore molecule that is engineered to contain two parts, with one part acting as the PeT donor and another part acting as the fluorophore. Electron transfer from the PeT donor to the excited fluorophore diminishes the fluorescence signal, and cleavage of the PeT donor leads to full activation of the fluorophore. In reality, some activatable probes are designed on the basis of two or more mechanisms of fluorescent quenching and activation (8). The activatable probes published to date can be classified into two types based on the location of fluorophore activation (2). One type involves enzymatic activation of the probes through cleavage by the extracellular or cell surface enzymes. This type of probe produces fluorescent signal primarily in the extracellular space. Another type of probe, known as a target cell–specific activatable probe, is quenched until activated through the lysosomal processing within targeted cells. Therefore, this type of probe generates fluorescent signal within the target cells. Regardless of the types, an important parameter that must be optimized for many activatable probes is the number of conjugated fluorophores per single mAb molecule. The number of conjugated fluorophores not only determines whether the fluorescence is “always on” or “silent,” but it also influences the binding specificity and pharmacokinetics (1, 2). Sano et al. labeled two different antibodies, Pan and Tra, with activatable near-infrared Alexa680 and ICG, respectively (1). Pan is a fully human IgG2 mAb against the extracellular domain of human epidermal growth factor receptor (EGFR, HER1), and Tra is a recombinant humanized mAb against the human EGFR type 2 (HER2). The two fluorescent dyes emit light at different wavelengths (650/702 nm and 780/820 nm for Alexa680 and ICG, respectively). The investigators administered the two activatable antibodies as a cocktail to mice bearing tumor xenografts and demonstrated the feasibility of multicolor target-specific fluorescence imaging with reduced background noise (1).