Abstract.SignificanceFluorescently guided minimally invasive surgery is improving patient outcomes and disease-free survival, but biomarker variability hinders complete tumor resection with single molecular probes. To overcome this, we developed a bioinspired endoscopic system that images multiple tumor-targeted probes, quantifies volumetric ratios in cancer models, and detects tumors inex vivosamples.AimWe present a new rigid endoscopic imaging system (EIS) that can capture color images while simultaneously resolving two near-infrared (NIR) probes.ApproachOur optimized EIS integrates a hexa-chromatic image sensor, a rigid endoscope optimized for NIR-color imaging, and a custom illumination fiber bundle.ResultsOur optimized EIS achieves a 60% improvement in NIR spatial resolution when compared to a leading FDA-approved endoscope. Ratio-metric imaging of two tumor-targeted probes is demonstrated in vials and animal models of breast cancer. Clinical data gathered from fluorescently tagged lung cancer samples on the operating room’s back table demonstrate a high tumor-to-background ratio and consistency with the vial experiments.ConclusionsWe investigate key engineering breakthroughs for the single-chip endoscopic system, which can capture and distinguish numerous tumor-targeting fluorophores. As the molecular imaging field shifts toward a multi-tumor targeted probe methodology, our imaging instrument can aid in assessing these concepts during surgical procedures.