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 in vivo, 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 in vivo 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
ex vivo nerve specificity, Oxazine 4 ex vivo nerve specific fluorescence has been difficult to visualize. In the present
study, we examined each step of the ex vivo fluorescence microscopy sample preparation procedure to discover how in
vivo nerve-specific fluorescence is changed during ex vivo 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 ex vivo was determined, which can be utilized
for visualization by fluorescence microscopy.
Connor W. Barth and Summer L. Gibbs, "Visualizing Oxazine 4 nerve-specific fluorescence ex vivo in frozen tissue sections," Proc. SPIE 9696, Molecular-Guided Surgery: Molecules, Devices, and Applications II, 96960R (Presented at SPIE BiOS: February 14, 2016; Published: 4 March 2016); https://doi.org/10.1117/12.2214204.
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