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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 in vivo neuronal tracing set-up. Methods: Neuronal tracing was achieved using a wheat germ agglutinin (WGA) lectin . functionalized with an azide-containing Cy5 dye ( N 3 -Cy5-WGA ). A Cy7 quencher dye ( Cy7-DBCO ) was subsequently used to yield Cy7-Cy5-WGA , a compound wherein the Cy5 emission is quenched by Förster resonance energy transfer to Cy7. The photophysical properties of N 3 -Cy5-WGA and Cy7-Cy5-WGA were evaluated together with deactivation kinetics in situ, in vitro (Schwannoma cell culture) , ex vivo (muscle tissue from mice; used for dose optimization), and in vivo ( nervus ischiadicus in THY-1 YFP mice) .  Results:  In situ , conjugation of Cy7-DBCO to N 3 -Cy5-WGA resulted in &gt;90% reduction of the Cy5 fluorescence signal intensity at 30 minutes after addition of the quencher. In cells, pretargeting with the N 3 -Cy5-WGA lectin yielded membranous staining, which could efficiently be deactivated by Cy7-DBCO over the course of 30 minutes (91% Cy5 signal decrease). In ex vivo muscle tissue, administration of Cy7-DBCO at the site where N 3 -Cy5-WGA was injected induced 80-90% quenching of the Cy5-related signal after 10-20 minutes, while the Cy7-related signal remained stable over time. In vivo,  Cy7-DBCO 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. Conclusion: The presented pretargeted fluorescence-quenching technology allowed fast and effective reduction of the background signal at the injection site, while preserving in vivo 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.

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