Background <p>Near-infrared fluorescence (NIRF) imaging assists surgeons intraoperatively to achieve radical resection of malignant tissue with one centimeter depth and can be supplemented with photoacoustic imaging to increase depth-of-view. Tumor-associated carbohydrate antigens are promising targets for tumor imaging with potential advantages over protein targeting. This study preclinically evaluates the anti-glycan tracers CH88.2-800CW (anti-Le^a/c/x) and CH129-800CW (anti-sdi-Le^a) for bimodal NIRF/PA imaging of gastrointestinal cancers.</p> Results <p>Using immunohistochemistry, we found that Le^a/c/x and sdi-Le^a were highly expressed in gastric and colorectal cancer tissue, with limited expression in healthy surrounding tissue, except for strong Le^a/c/x expression in healthy colorectal epithelium. Bimodal NIRF/PA imaging using CH88.2-800CW and CH129-800CW was performed on subcutaneous and orthotopic HT-29_luc2 (colon cancer) and BxPC-3_luc2 (pancreatic cancer) tumor-bearing mice, using rituximab-800CW as a negative control tracer. At 96&#xa0;h post-injection, all orthotopic tumors were delineated using the clinical Artemis NIRF imager with mean CH88.2-800CW and CH129-800CW tumor-to-background ratios of 4.8 ± 1.4 and 4.9 ± 0.5 for the HT-29_luc2 model, and 2.5 ± 0.3 and 2.9 ± 0.4 for the BxPC-3_luc2 model, respectively. Similarly specific photoacoustic signal was observed within all tumors for both CH88.2-800CW and CH129-800CW. Biodistribution analyses showed high tumor fluorescence with minimal signal in healthy organs, including the liver and kidneys.</p> Conclusions <p>Bimodal NIRF/PA imaging employing CH88.2-800CW and CH129-800CW facilitates real-time, high-contrast gastrointestinal tumor visualization. Given their strong and mostly tumor-specific expression, both tracers hold promise as effective imaging agents for gastrointestinal cancers, and are compelling candidates for further clinical evaluation.</p>