The outcomes of oncologic surgery critically depend on complete removal of the tumor mass, which prevents tumor regrowth and disease reoccurrence. This is a challenging task, especially in cases of invasive and disseminated disease in which tumor margins are poorly defined and microscopic tumors are often indistinguishable from surrounding normal tissues. Preoperative diagnostic imaging provides a wealth of information on the tumors' location and size. However, during surgery, surgeons identify tumors and tumor margins largely by visual inspection of the surgical field and by palpation; as a result, tumors often remain undetected, and tumor margins are determined incorrectly. On the other hand, excessive resection of tumor margins often leads to severe postoperative morbidity. These problems may be solved using real- time intraoperative visualization of tumors enabled by near-infrared fluorescence (NIRF), which would give a surgeon the ability to locate and remove the tumors that cannot be seen by the naked eye and enable more precise determination of tumor margins. The objective of this pilot project is to demonstrate the feasibility of sensitive and accurate intraoperative tumor visualization by developing and testing in an animal model of cancer a new generation of tumor-targeted NIRF probes. Our approach is based on the hypothesis that such advanced probes can be developed using a novel class of rationally engineered molecules-designed ankyrin repeat proteins (DARPins)-as carriers of NIR fluorophores. Owing to their small size, high affinity and specificity for tumor biomarkers, DARPins rapidly and efficiently accumulate in tumors and clear from nontarget tissues. DARPins are easy to produce in bacteria and purify; they can be easily modified genetically to enable precise, site-specific conjugation with fluorophores. DARPins' highly stable core structure results in prolonged stability in human blood. These properties make DARPins an excellent alternative to antibodies (Ab), Ab derivatives, peptides, and small molecules, which are traditionally used for imaging probe development. We will test our hypothesis and achieve our objective by pursuing two specific aims: Specific Aim 1. Develop and characterize in vitro DARPin-based NIRF probes that target human epidermal growth factor receptor 2 (Her2). Specific Aim 2. Demonstrate the feasibility of intraoperative visualization of disseminated tumors by using DARPin-derived NIRF probes in a mouse model of ovarian cancer. The outcomes of this feasibility project will have a significant positive impact on cancer management by making surgical resection of tumors more efficient and reducing post-surgical morbidity, thereby prolonging and saving human life.