Abstract Lymph node metastasis is the most significant predictor of survival for patients with many cancers including breast cancer and head and neck squamous cell carcinoma (HNSCC). The gold standard for detecting metastatic disease is lymph node dissection where all regional lymph nodes are removed and histologically analyzed. This procedure has significant morbidity so clinicians use indirect methods to detect involved nodes such as imaging, and sentinel node biopsy (SNB). The long-term goal of this project is to establish tumor-activatable near infrared (NIR) nanoprobes to delineate cancer involved nodes to allow surgical removal while preserving normal structures during surgery without relying on indirect methods which have a significant error rate. We will capitalize on our recent invention of pH transistor nanoprobes (PTN) to target acidic tumor pH, a ubiquitous hallmark of cancer. PTN like an electronic transistor has a binary all or nothing response, in this case to the tumor microenvironment fluorescently turning completely on in tumors and remaining completely off in normal tissue. This all or nothing behavior is critical for detecting the small volume disease in lymph nodes. We adopted the PTN design to indocyanine green (I-PTN), an FDA-approved NIR dye with a sharp pH response (?pHON/OFF = 0.15 pH). Using a SPY Elite clinical camera, we have shown the effectiveness of I-PTN nanoprobes in the visualization of various solid cancers as well as cancer involved lymph nodes. Preliminary data show accurate identification of cancer involved lymph nodes in vivo allowing precise surgical removal. In the proposed project, we will specifically optimize PTN activation for detecting metastatic cancer in lymph nodes. We will test the central hypothesis that I-PTN-guided removal of cancer involved lymph nodes improves survival and without the need for removal of all regional nodes. To test our hypothesis, we will carry out the following specific aims: 1) Establish a series of fine-tuned ICG-PTN nanoprobes (pH 5.4 to 7.1) with 0.15 pH increments and determine the optimal pH for detecting HNSCC and breast cancer in nodal metastases from orthotopic xenographs 2) Evaluate the accuracy of ICG-PTN nanoprobes in the delineation of tumor involved nodes versus uninvolved nodes. We will harvest at risk nodes in mice bearing tumors and determine the sensitivity and specificity of pH transistor nanoprobes for detecting nodal metastases. 3) Identify and remove cancer involved lymph nodes and primary tumors to investigate the long-term survival benefit of ICG-PTN-guided resection.