Visualizing Oxazine 4 nerve-specific fluorescence in frozen tissue sections.

Nerve damage plagues surgical outcomes and remains a major burden for patients, surgeons, and the healthcare system. Fluorescence image-guided surgery using nerve specific small molecule fluorophores offers a solution to diminish surgical nerve damage through improved intraoperative nerve identification and visualization. Oxazine 4 has shown superior nerve specificity in initial testing , while exhibiting a red shifted excitation and emission spectra compared to other nerve-specific fluorophores. However, Oxazine 4 does not exhibit near-infrared (NIR) excitation and emission, which would be ideal to improve penetration depth and nerve signal to background ratios for imaging. Successful development of a NIR nerve-specific fluorophore will require understanding of the molecular target of fluorophore nerve specificity. While previous small molecule nerve-specific fluorophores have demonstrated excellent nerve specificity, Oxazine 4 nerve specific fluorescence has been difficult to visualize. In the present study, we examined each step of the fluorescence microscopy sample preparation procedure to discover how nerve-specific fluorescence is changed during tissue sample preparation. Through step-by-step examination we found that Oxazine 4 fluorescence was significantly diminished by washing and mounting tissue sections for microscopy. A method to preserve Oxazine 4 nerve specific fluorescence was determined, which can be utilized for visualization by fluorescence microscopy.