Abstract. To improve surgical guidance toward prostate draining lymph nodes, we investigate the potential of intraoperative fluorescence imaging and combined pre-and intraoperative multimodality imaging approaches. Transgenic adenocarcinoma mouse prostate mice with spontaneous prostate tumors are injected intratumorally with: 1. a cocktail of patent blue (Pb) and indocyanine green (ICG); 2. a cocktail of albumin radiocolloids ( 99m TcNanoColl), Pb, and ICG; or 3. a cocktail of radiolabeled albumin ( 99m Tc-Vasculosis), Pb, and ICG. The distribution of these imaging agents over the lymph nodes (LNs) are studied at different time points after injection. We find that at 60-min postinjection, ICG significantly improves the detection of the LNs compared to Pb, 53 versus 7%, respectively. Moreover, a cocktail of ICG and 99m Tc-NanoColl improves the fluorescent detection rate to 86%, equalling that of the clinically applied 99m Tc-NanoColl. A similar overlap is observed in our initial clinical pilot data. Fluorescent detection of the LNs using a ICG with 99m Tc-Vasculosis gives similar results as "free" ICG (58%; 60 min). A 99m Tc-NanoColl, Pb, and cocktail ICG enriches the standard 99m Tc-NanoColl approach by adding optical detection of the sentinel lymph nodes. Furthermore, this approach improves fluorescent-based guidance and enables both accurate surgical planning and intraoperative detection, based on a single injection. C 2011 Society of Photo-Optical Instrumentation Engineers (SPIE).