Full Text | PDF

Theranostics 2020; 10(21):9890-9898. doi:10.7150/thno.46806 This issue Cite

Research Paper

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

Tessa Buckle^1✉, Steffen van der Wal¹, Danny M. van Willigen¹, Germaine Aalderink¹, Gijs H. KleinJan^1,2, Fijs W.B. van Leeuwen¹

1. Interventional Molecular Imaging Laboratory, Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands.
2. Department of Urology, Leiden University Medical Center, Leiden, The Netherlands.

✉ Corresponding author: T. Buckle, Interventional Molecular Imaging Laboratory, Department of Radiology; Leiden University Medical Center, Albinusdreef 2 (C2-S zone); PO BOX 9600, 2300 RC Leiden, The Netherlands. E-mail: t.bucklenl.

Abstract

Introduction: 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 (N₃-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 N₃-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 N₃-Cy5-WGA resulted in >90% reduction of the Cy5 fluorescence signal intensity at 30 minutes after addition of the quencher. In cells, pretargeting with the N₃-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 N₃-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.

Keywords: Pretargeting, fluorescence imaging, nerve imaging, image-guided surgery, fluorescence activation

Citation styles

©2024 Ivyspring International Publisher. Terms of use