Nile Red derivatives enable improved ratiometric imaging for nerve-specific contrast

Abstract. Surgical nerve damage due to difficulty with identification remains a major risk for postsurgical complications and decreased quality of life. Fluorescence-guided surgery offers a means to specifically highlight tissues of interest such as nerves and a number of fluorescence-guided surgical systems are in clinical trial or are approved for clinical use. However, no clinically approved nerve-specific fluorophores exist. In addition, many preclinical nerve-specific fluorophores tend to accumulate in adipose tissue due to the molecular composition similarities between the two tissues, making it challenging to generate a specific nerve signal. To alleviate this difficulty, we have synthesized a library of oxazine fluorophores based on the Nile Red scaffold, with the goal of strong adipose specificity without nerve uptake to facilitate ratiometric imaging. The library was screened for tissue specificity ex vivo and in vivo, enabling quantification of adipose-, nerve- and muscle-specific uptake as well as selection of the best candidate for adipose selectivity without nerve signal. We showed our selected Nile Red fluorophore improved nerve contrast using ratiometric imaging, especially nerve-to-adipose contrast as compared to the parent Nile Red compound or nerve-specific imaging alone. This adipose-specific Nile Red derivative could be used in future fluorescence-guided surgery applications where adipose- or nerve-specific contrast is required.