ABSTRACT Breast cancer frequently metastasizes to the axillary lymph nodes (ALN). The cancer spread from the primary breast tumor can occur during the early stages, and ALN metastasis is usually the earliest detectable cancer spread. Sentinel lymph node (SLN) biopsy is the standard approach for axillary staging in breast cancer patients with no palpable axillary adenopathy or when ALN metastasis is not detected during the presurgical diagnosis. Currently, SLN is identified by a peritumoral injection of a radioactive tracer, technetium 99mTc and/or blue dye, followed by a SLN biopsy for pathological examination. Although this peritumoral injection method can identify the location of SLN, it does not intraoperatively distinguish between LN metastases and healthy LN as it stains SLN regardless of the status of LN metastases. Thus, once SLN is identified by 99mTc and/or blue dye, SLN is surgically removed regardless of cancer metastases status in SLN, and sent for pathological assessment. The majority of patients with breast cancer (~70%) who undergo a SLN biopsy are pathologically negative. Although the clinical benefits of a SLN biopsy have been observed, SLN biopsies are often associated with postoperative complications such as lymphedema, seroma formation, sensory nerve injury, and limitation in range of motion. Moreover, after a SLN biopsy, if pathological reports show cancer negative in the SLN, then a complete ALN dissection can be avoided. When SLN is cancer positive, patients need a second operation to complete the ALN dissection. This decision can be made during the primary tumor resection or SLN biopsy if surgeons can detect LN involvement intraoperatively. Therefore, a new intraoperative method for a clinically translated surgical visualization tool that can accurately detect LN involvement is clinically needed to eliminate any delay in treatment and prevent unnecessary surgeries. To overcome previous and current challenges in identifying LN with cancer cells, our approach in this application aims to intraoperatively distinguish LN with cancer cells from normal LN with a real-time visualization tool. Our preliminary results with one breast cancer cell line showed that ICG-p28 preferentially accumulated at the primary breast tumor and LN metastases, but not in healthy LN. Based on our preliminary data, we hypothesize that our real-time imaging approach with ICG-p28 can intraoperatively/accurately distinguish between LN metastases and healthy LN which will substantially improve the health of breast cancer patients. We will test our hypothesis in mimicked intraoperative settings. Each aim/sub-aim will occur in the collaborative and interdisciplinary environment to carry out our proposed research. Our unique imaging approach can potentially provide a significant impact on SLN biopsy procedures. It will potentially provide better treatments for breast cancer patients, which is a major milestone and relevant to the NIH focus area.