Epidermal growth factor (EGF) is a 53-amino acid cytokine (6.2 kDa) secreted by ectodermic cells, monocytes, kidneys and duodenal glands (1). EGF stimulates growth of epidermal and epithelial cells. EGF with at least seven other growth factors and their transmembrane receptor kinases play important roles in cell proliferation, survival, adhesion, migration and differentiation. The EGF receptor (EGFR) family consists of four transmembrane receptors, including EGFR (HER1/erbB-1), HER2 (erbB-2/neu), HER3 (erbB-3) and HER4 (erbB-4) (2). HER1, HER3 and HER4 comprise three major functional domains: an extracellular ligand-binding domain, a hydrophobic transmembrane domain and a cytoplasmic tyrosine kinase domain. No ligand has been clearly identified for HER2. However, HER2 can be activated as a result of ligand binding to other HER receptors with the formation of receptor homodimers and/or heterodimers (3). HER1 as well as HER2 are overexpressed on many solid tumor cells such as breast, non-small-cell lung, head and neck, and colon cancer (4-6). The high levels of HER1 and HER2 expression on cancer cells are associated with a poor prognosis (7-10). Optical fluorescence imaging is increasingly used to monitor biological functions of specific targets (11-13). However, the intrinsic fluorescence of biomolecules poses a problem when fluorophores that absorb visible light (350-700 nm) are used. Near-infrared (NIR) fluorescence (700-1,000 nm) detection avoids the background fluorescence interference of natural biomolecules, providing a high contrast between target and background tissues. NIR fluorophores have a wider dynamic range and minimal background as a result of reduced scattering compared with visible fluorescence detection. They also have high sensitivity, resulting from low fluorescence background, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is becoming a non-invasive complement to radionuclide imaging in small animals. Trastuzumab is a humanized IgG monoclonal antibody (MAb) against the extracellular domain of recombinant HER2 with an affinity constant () of 0.1 nM (14). Cardiotoxicity is the most serious complication of using trastuzumab in humans with breast cancer (15). One potential application of a radiolabeled anti-HER2 MAb is the pretreatment imaging of breast cancer patients to predict the therapeutic efficacy of trastuzumab. In-Trastuzumab and Ga-F(ab')-trastuzumab have been developed for imaging of human breast cancer (16-19). Trastuzumab has also been successfully coupled with Cy5.5 NIR dye for optical imaging of HER2 density in tumors in mice (20).