Inflammation is a major unifying syndrome associated with a variety of injury and diseases. When severe, inflammatory diseases can endanger and even kill patients. Direct diagnosis of the extent of inflammatory responses is often made histological with an invasive biopsy. Furthermore, multiple biopsies are often required to assess the extent of inflammatory reactions in a large wound. The proposed work is aimed at developing a new non-invasive method to assess the extent of inflammatory responses in real-time in vivo. Reactive oxygen species (ROS) released by polymorphonuclear neutrophils have been shown to be a good indicator of the degree of inflammatory reactions. Several ROS-reactive bioluminescent probes have been investigated in recent years to detect ROS activities in vivo. Unfortunately, these probes alone cannot be used to quantify the degree of inflammatory responses due to the fact that the extent of the bioluminescent signals is also probe-concentration dependent. To address this challenge, we adopt ratiometric probe technique in which both ROS-sensitive agents and ROS-insensitive reference dye are conjugated to biocompatible particle carriers. The bioluminescent/reference fluorescent intensity ratios can be calculated to reflect the extent of localized ROS activities while circumventing the variations of bioluminescent intensities associated with the ROS probe concentrations. Our preliminary studies have led to the development of ROS ratiometric probes which can reliably detect localized ROS activities in vivo and inflamed tissue in the wound site. Based on these exciting observations, the proposed work will create a series of ROS ratiometric probes to detect ROS activities in vitro (Aim 1) and to assess the extent of inflammatory responses mediated by different injuries or inflammatory stimuli in vivo (Aim 2). Finally, since all commercial imagers are designed with an enclosed chamber only suitable for cell and small animal imaging, a handheld imager for ROS sensing in large animals and human will be developed as part of this investigation. The successful completion of this proposed work will help the development of a new diagnosis tool which can be applied to non-invasively monitor the inflammatory processes in real-time and assist the development of new treatments to effectively reduce inflammation and improve wound healing reactions. PUBLIC HEALTH RELEVANCE: A series of novel reactive oxygen species (ROS) ratiometric imaging probes and a hand-held optical device will be fabricated to monitor and quantify in situ ROS activities and associated inflammatory responses in vivo. The successful completion of the proposed work will help the development of a new diagnosis tool which can be effectively used to non-invasively assess the extent of inflammatory processes in real-time and substantially improve the quality of patient care.