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with the lower limbs directly exposed to cold. 0.1 ml of 1 mg/ml L-012 probe was injected (i.p.) to determine the initial changes in ROS activity. Noninvasive luminescence and X-ray images obtained show robust increase in ROS activity at BAT sites which is generally accumulated at the regions between the scapulae. Apart from the brown fat depot sites, increase in ROS activity was also observed at additional sites that include eyes. These real-time monitoring methodologies of ROS activity detection by in vivo imaging will greatly enhance the understanding of the early phase response to cold stress and aid in development of intervention strategies in several physiological and pathological processes.