Conjugation of the optical reporter, a cyanine dye, to tilmanocept proceeded in a manner previously described.24 The NIR dye was covalently coupled to the amino-terminated leashes on the dextran backbone of DTPA-mannosyl-dextran (tilmanocept, Navidea Biopharmaceuticals, Ohio). The mean number of DTPA, mannose, and amines per dextran of the tilmanocept preparation was 4.4, 16.8, and 5.5, respectively. The mean molecular weight was and Stokes radius was 7.7 nm. Conjugates with different IRDye 800CW to tilmanocept ratios were prepared. Briefly, tilmanocept (0.25 μmol) was combined with varying amounts (molar excess of 1 to 10 equivalents) of IRDye 800CW-NHS-ester (LI-COR Biosciences, Nebraska) in 1.1 ml dimethyl sulfoxide. After brief vortexing, the mixture was incubated overnight on a rolling rotor at 30°C. After diluting the reaction mixture with deionized water, IRDye 800CW-tilmanocept (800CW-tilmanocept) was purified by centrifuging the product through an ultrafiltration system (Ultra 15, Amicon Corp., Maryland, 3 kDa molecular weight cut-off). After five times of centrifugation, deionized water was added to the concentrated retentate and a sample was removed for analysis of purity. The centrifugation was repeated until no free IRDye 800CW could be detected in the retentate as measured by high-performance liquid chromatography (HPLC) with fluorescence detection. The product was lyophilized, weighed, and redissolved in phosphate-buffered saline (PBS) to form a stock solution with a final concentration of . Purity of the final product was confirmed by size exclusion (TSKgel G2000SWxl, Tosoh Bioscience LLC, Tokyo, Japan) HPLC (System Gold, Beckman-Coulter, California) using 0.9% saline as the mobile phase. The eluate was monitored by UV absorbance and fluorescence (L-7480, Hitachi America, Ltd., Michigan).