Fluorescence background quenching as a means to increase Signal to Background ratio - a proof of concept during Nerve Imaging.

Adequate signal to background ratios are critical for the implementation of fluorescence-guided surgery technologies. While local tracer administrations help to reduce the chance of systemic side effects, reduced spatial migration and non-specific tracer diffusion can impair the discrimination between the tissue of interest and the background. To combat background signals associated with local tracer administration, we explored a pretargeting concept aimed at quenching non-specific fluorescence signals. The efficacy of this concept was evaluated in an neuronal tracing set-up. Neuronal tracing was achieved using a wheat germ agglutinin (WGA) lectin functionalized with an azide-containing Cy5 dye (). A Cy7 quencher dye () was subsequently used to yield , a compound wherein the Cy5 emission is quenched by Förster resonance energy transfer to Cy7. The photophysical properties of and were evaluated together with deactivation kinetics (Schwannoma cell culture) (muscle tissue from mice; used for dose optimization), and ( in THY-1 YFP mice), conjugation of to resulted in >90% reduction of the Cy5 fluorescence signal intensity at 30 minutes after addition of the quencher. In cells, pretargeting with the lectin yielded membranous staining, which could efficiently be deactivated by over the course of 30 minutes (91% Cy5 signal decrease). In muscle tissue, administration of at the site where was injected induced 80-90% quenching of the Cy5-related signal after 10-20 minutes, while the Cy7-related signal remained stable over time. effectively quenched the non-specific background signal up to 73% within 5 minutes, resulting in a 50% increase in the signal-to-background ratio between the nerve and injection site. The presented pretargeted fluorescence-quenching technology allowed fast and effective reduction of the background signal at the injection site, while preserving nerve visualization. While this proof-of-principle study was focused on imaging of nerves using a fluorescent WGA-lectin, the same concept could in the future also apply to applications such as sentinel node imaging.