Project Summary: Tissue necrosis is a form of cell death caused by a wide variety of diseases and injuries. Current methods of detecting tissue necrosis to guide surgical decision making are limited. In burn injury, clinical visualization of tissue necrosis is the standard of care; however, it is an imprecise method that can result in delays in care, unnecessary surgery, and removal of viable tissue. There is a critical need to identify novel methods to improve the detection of necrosis in burn injury to aid perioperative clinical decision making. While Indocyanine Green Angiography (ICGA) has been shown to identify burn depth using perfusion as a surrogate marker for necrosis, it has not been widely adopted for clinical decision making. Recently, clinical trials using delayed imaging of high dose ICG (Second Window Indocyanine Green - SWIG) have shown promise in image-guided surgical resection of tumors. We propose that combined imaging with ICGA and SWIG can be employed to enhance surgical decision-making in burn injury as well as in many disease processes involving necrosis. The knowledge gained from this project will fill the critical need to prevent unnecessary surgery, improve surgical precision, and provide insight into ICG localization in inflamed and necrotic tissue. The goal of this project is to characterize the ICGA and SWIG fluorescence in burn inflammation and necrosis on a macroscopic and microscopic level. Specific Aim 1 will characterize fluorescent signals from ICGA and SWIG in the healing potential of indeterminate depth burns in humans. Specific Aim 2 will evaluate the diagnostic accuracy of intraoperative fluorescence-guided surgical resection of necrotic tissue in humans. Specific Aim 3 will characterize ICG fluorescence quantification in inflamed, necrotic, and healthy tissues and determine substrate localization using cell culture and animal models. To attain our goal, we will use a team science approach including a burn surgeon scientist who has extensive experience in human thermal injury models and clinical expertise in the surgical care of burn patients along with imaging experts who have a track record for developing advanced fluorescence-based technologies for in vivo imaging, including a surgical imaging technology called “transient lighting” that allows simultaneous white light and low-level fluorescence visualization in ambient lighting conditions. Transient lighting is especially critical in burn surgery to augment the visualization of the wound with ICG fluorescence under full white lighting. This project will result in preclinical and clinical data testing the use of ICG for direct detection of necrotic tissue using a fluorescence imaging device optimized for burn surgery, while developing a platform for quantification of tissue necrosis and characterization of ICG-avid necrosis. These studies will provide necessary data to inform the design of a larger clinical trial to determine the efficacy and validity of...