Humanized monoclonal antibodies (MAbs) conjugated to radionuclides used for the detection and treatment of cancers have limited efficacy because they are large glycoprotein molecules (~150 kD) that cannot penetrate deep into cancerous tumors (1). In general, radioimmunoconjugates, including those used for imaging purposes, have a prolonged blood circulation and, if used for radioimmunotherapy ( high-energy β-emitters such as yttrium or rhenium, etc., or α-emitters such as bismuth or astatine, etc.), can impair the functioning of normal cells and may be myelotoxic (1). The use and limitations of radioimmunoconjugates as imaging and therapeutic agents were discussed in detail by Sharkey and Goldenberg (1). As an alternative to using radionuclides in conjunction with MAbs for the detection of cancer, investigators have explored the use of fluorescent agent-MAbs and other molecule conjugates for the preclinical imaging of cancerous tumors (2-6). However, the fluorescent dye-MAb probes, although cheap to generate, have blood circulation and tumor penetration drawbacks similar to the radioimmunoconjugates and also exhibit a high background because of autofluorescence generated in the tissue (6, 7). To circumvent problems observed with the optical probes, investigators have generated self-quenching, targeted fluorescent dye-MAb conjugates that are non-fluorescent in the native state but generate a signal only when activated by degradation of the targeting MAb or molecule by a specific cellular process or organelle (8). Accumulation of the fluorescent dye within a target cell would reduce the non-specific background and decrease the blood pool signal. The chemical structure of self-quenching probes is such that a quencher molecule is closely associated in alignment with the fluorophore. Enzymatic cleavage of the structure results in separation of the quencher from the fluorophore and in the generation of a fluorescent signal (8). Near-infrared (NIR) probes such as Alexa fluor and Cy5.5 are favored over other optical agents because they exhibit superior tissue penetration (hemoglobin and water have low absorption of the NIR signal at the operating wavelengths of these probes), and when two or more NIR probe molecules are conjugated, the fluorescent signal quenches itself. Cleavage of the conjugation bonds results in fluorescence dequenching and generation of a signal that is suitable for imaging (9-11). However, it is pertinent to mention that only a specific number of the NIR probe molecules can be conjugated to a macromolecule without changing the structural and pharmacokinetic properties of the macromolecule. Ogawa et al. conjugated multiple copies of Alexa fluor 680 (Alexa680) to trastuzumab (Tra), a humanized MAb that specifically targets the human epidermal growth factor receptor 2 (HER2), to synthesize a self-quenching, targeted NIR probe (Tra-Alexa680(SQ)) (12). Tra is approved by the United States Food and Drug Administration for use as a single agent or an adjuvant to treat certain types of breast cancer and is being tested in clinical trials for the treatment of other neoplasms in humans. A characteristic feature of HER2 is that, upon binding a ligand, including the MAb directed against it, the receptor-ligand complex is internalized by the cell for enzymatic digestion in the lysosomes (13, 14). Proteolytic digestion of the receptor and the bound MAb in the lysosome releases the quencher from the fluorophore, resulting in generation of a signal. Therefore, a fluorophore that is “off” in the extracellular environment is turned “on” in an intracellular location. Ogawa et al. investigated the receptor specificity of Tra-Alexa680(SQ) under conditions using NIH-3T3 cells transfected with the HER2 gene (3T3/HER2 cells), and to study the characteristics of the fluorophore-MAb conjugate the investigators used nude mice bearing xenograft and orthotropic tumors generated with 3T3/HER2 or BALB/3T3/HER2 cells (12).