Our attention then turned to two-photon imaging of ONOO -and ATP using ATP-LW, with the same cell models used above. The results (Fig. ) confirm the ability of ATP-LW to image both ONOO -and ATP, using an excitation of 976 nm for the former, and 1028 nm for the latter. Since the power for excitation at 1028 nm for a two-photon microscope is lower, we extended the emission range for the red channel (i.e. 575-680 nm). for cellular imaging. Importantly, using ATP-LW we have been able (using two different channels) to monitor in real-time for the first time the enhancement of ONOO -and depletion of ATP during APAP-induced hepatotoxicity. Moreover, we have illustrated the signaling pathways for APAP-induced toxicity where ONOO -increase and ATP depletion are responsible for hepatic necrosis. We anticipate that ATP-LW can be extended for imaging the important fluctuations of these two biomarkers in other diseases such as ischemia-reperfusion injury.