Changes in the UV-Vis absorption of ATP-LW in the presence of ONOO -and ATP were investigated. The addition of ONOO -produced a new absorption peak at 445 nm which is consistent with the intramolecular charge transfer (ICT) process seen with 4-hydroxy-1,8naphthalimide product (Fig. ). Similarly, the addition of ATP to ATP-LW resulted in an increase in absorption at 533 nm. This is indicative of the ATP-induced opening of the spirolactam ring found on the rhodamine fluorophore (Fig. ). ATP-LW was initially non fluorescent, due to the quenching effects of the boronic ester on the 1,8-naphthalimide unit and ring-closed rhodamine being non-fluorescent. Upon the addition of ONOO -(0-16 μM), a significant fluorescence increase at 562 nm/568 nm was observed when excited at 450/488 nm (Fig. , Fig. ). 488 nm excitation wavelength was used for measurements in aqueous solution since it was the closest available excitation wavelength for cellular imaging experiments (green channel, λem = 500-575 nm, λex = 488 nm). Minimal fluorescence increase was observed for light excitation at 520 nm upon the addition of ONOO -(0-16 μM) (Fig. ).