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-backgroundin vitroand 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.