Surgery is the preferred treatment option for most solid
tumors. However, inaccurate detection of
cancer borders leads to either incomplete removal of malignant cells or excess excision of healthy tissue. While fluorescent
contrast agents and imaging systems improve
tumor visualization, they can suffer from low signal-to-background and are prone to technical artifacts. Ratiometric imaging has the potential to eliminate many of these issues such as uneven probe distribution, tissue autofluorescence, and changes in positioning of the light source. Here, we describe a strategy to convert quenched
fluorescent probes into ratiometric
contrast agents. Conversion of the
cathepsin-activated probe, 6QC-Cy5, into a two-fluorophore probe, 6QC-RATIO, significantly improved signal-to-background in vitro and in a mouse subcutaneous
breast tumor model.
Tumor detection sensitivity was further enhanced using a dual-substrate AND-gate ratiometric probe, Death-Cat-RATIO, that fluoresces only after orthogonal processing by multiple
tumor-specific
proteases. We also designed and built a modular camera system that was coupled to the FDA-approved da Vinci Xi robot, to enable real-time imaging of ratiometric signals at video frame rates compatible with surgical workflows. Our results demonstrate that ratiometric camera systems and imaging probes have the potential to be clinically implemented to improve surgical resection of many types of
cancer.