Accidental nerve transection or injury is a significant morbidity associated with many surgical interventions, resulting in persistent postsurgical numbness, chronic pain, and/or paralysis. Nervesparing can be a difficult task due to patient-To-patient variability and the difficulty of nerve visualization in the operating room. Fluorescence image-guided surgery to aid in the precise visualization of vital nerve structures in real time during surgery could greatly improve patient outcomes. To date, all nerve-specific contrast agents emit in the visible range. Developing a nearinfrared (NIR) nerve-specific fluorophore is poised to be a challenging task, as a NIR fluorophore must have enough "double-bonds" to reach the NIR imaging window, contradicting the requirement that a nerve-specific agent must have a relatively low molecular weight to cross the blood-nervebarrier (BNB). Herein we report our efforts to investigate the molecular characteristics for the nervespecific oxazine fluorophores, as well as their structurally analogous rhodamine fluorophores. Specifically, optical properties, physicochemical properties and their in vivo nerve specificity were evaluated herein.