PROJECT SUMMARY The main treatment of cancer is surgery, which has shown the best overall survival rate compared to radiotherapy and chemotherapy; but after the surgery, detection of lymph nodes metastases strongly influences the patient prognosis. Moreover, the most important factor for predicting long-term cancer survival is completeness of the surgical resection; but this may be difficult to achieve with microscopic disease. Unfortunately, current imaging modalities to localize cancer, such as PET, CT, and MRI, which are used pre- and post-operatively, are limited by resolution - larger than 0.5–1cm to be detectable. Consequently, current treatment failure in cancer is partly driven by lack of molecular imaging modalities to assess occult metastasis, response to therapy, or improved surgical resection techniques. Thus, detection of micrometastatic disease, achieving good resection margins at the time of surgery, and proper identification of nodal spread remain hurdles to improving surgical outcomes, treatment morbidity, and overall survival outcomes for our patients. The proposed research aims to address the limitations of current imaging modalities in cancer detection and surgical resection. By developing near-infrared chemiluminescent and fluorescent (NIR CFL) chitosan-based nanoparticles (CNPs), we seek to improve the detection of micrometastatic disease and guide image-guided surgery. These CNPs can be activated by body heat and stored at low temperatures, providing convenience and safety during use. Through the conjugation of synthetic targeting ligands, we aim to enhance tumor targeting and assess surgical margins and metastatic lymph nodes using chemiluminescence and fluorescence optical imaging. Our approach combines in vivo and ex vivo imaging modes, enabling the identification of deep anatomical locations and microscopic targets with high contrast and excellent signal-to-noise ratio. Preliminary results obtained in mice demonstrate the feasibility and practicality of our approach, showcasing the ability of chemiluminescence imaging to detect target sites over 4cm below the body surface. The anticipated outcomes of this research include advancing the combined use of optical imaging in vivo, enabling improved detection of micrometastatic disease, reducing surgical morbidity by preserving normal tissue, and providing valuable guidance for management decisions in cancer patients. By addressing the challenges associated with cancer treatment failure, incomplete resection, and nodal spread identification, this project has the potential to significantly enhance surgical outcomes, reduce treatment-related complications, and improve overall survival rates for patients.