Optical imaging was then performed to identify nodal metastases. neck squamous carcinoma malignancy UM-SCC-22B cells or human ovarian malignancy SKOV-3 cells. The metastases within LNs Polyphyllin B were confirmed by bioluminescence imaging (BLI). IRDye800-Antibodies were intravenously administered 24 h before local administration of Cy5.5-HA. Optical imaging was then performed to identify nodal metastases. Results: Binding of HA with LYVE-1 was confirmed by ELISA and fluorescence staining. HA with a size of 10K was chosen based on the favorable migration and retention profile. After sequential administration of IRDye800-antibodies intravenously and Cy5. 5-HA locally to a mouse model with LN metastases and fluorescence optical Polyphyllin B imaging, partially metastasized LNs were successfully distinguished from un-metastasized LNs and fully tumor occupied LNs, based on the different transmission patterns. Conclusions: Fluorophore conjugated HA is usually a potential lymphatic mapping agent for SLNB. Dual-tracer imaging with the combination of lymphatic mapping brokers and tumor targeting brokers can identify tumor metastases within SLNs, thus may provide accurate and real-time intra-operative guidance to spare the time spent waiting for a biopsy result. Keywords: metastasis, sentinel lymph node, optical imaging, hyaluronic acid, EGFR, HER2. Introduction Due Polyphyllin B to the large cavity of lymph vessels and slow velocity of lymph, the lymphatic system serves as a transport route for disseminating tumor cells, resulting in metastases 1. Consequently, many types of malignant tumors such as breast malignancy, melanoma, and prostate malignancy are prone to metastasize first to regional lymph nodes (LNs), through tumor associated lymphatic channels 2, 3. Since sentinel lymph nodes (SLNs) are the nodes that receive Polyphyllin B direct lymphatic drainage from a primary tumor site 4, the status of SLNs serves as an indication of prognosis and therapeutic decision-making 5, 6. So far, the gold standard to stage the SLNs is usually lymphadenectomy and histological evaluation, which is usually invasive and limited by surgical field for nodal sampling and lack of accuracy 7. With the development of imaging techniques, currently, pre-surgical diagnosis of SLNs is usually often based on their morphologic change observed by magnetic resonance imaging (MRI) or x-ray computed tomography (CT) 8, 9. For intra-operative guidance, the most common strategy is usually to locally administer technetium-99m radiocolloid then a blue dye (either patent blue or methylene blue) several hours later. SLNs can be detected by a gamma probe as warm spots and visualized by bright field as blue nodes 10-13. Although the CLG4B value of this process has been substantiated in numerous clinical studies 11, 12, it has several drawbacks. Radiocolloid requires complicated and restrictive radioactive management norm and scintigraphy or gamma probes suffer from low sensitivity and poor spatial resolution. The blue dye may stain the surgery area into blue, which may cause inconvenience for the surgery 14. Moreover, these blue dyes are of small molecular size and migrate through lymphatic system rapidly, giving rise to poor accumulation in the SLN and mess up SLNs with LNs of secondary echelon 15. Recently, intraoperative fluorescent imaging provided a promising option for mapping SLNs. Other than the combination of radiocolloid and blue dyes, fluorescence dyes are radio-free, relatively tissue penetrable and visual view clean. For example, indocyanine green (ICG), a FDA approved near-infrared (NIR) dye with good safety profile, has been intensively investigated both preclinically and clinically for SLN mapping 16, 17. Similar to the patent blue or methylene blue dye, ICG is also reported as fast migration and with low retention time in SLNs. One major limitation of the currently available method is incapable of differentiating SLNs with or without tumor metastases. In most cases, both the doctor and patient have to wait at least 40 moments for the result of pathological examination. Thus, an ideal lymphatic imaging strategy should have high transmission background ratio for obvious SLN detection, be able to differentiate tumor metastasis and provide real-time.