A variety of topical and systemic treatments exist to treat the chronic wounds and burns that afflict over 6 million patients annually in the U.S. However, wound infections/biofilms and necrotic tissue often exacerbate healing times and ultimately contribute to > $25 billion in added annual U.S. health care costs. Generally, wounds that fail to resolve are subjected to repeated painful debriding protocols to remove infected and decaying tissues in order to stimulate proper healing. Development of an effective, low cost, portable wound debridement treatment could improve patient outcomes, reduce pain, and ultimately improve health care costs. This proposed project seeks to develop a cold, atmospheric pressure (CAP) plasma scalpel to selectively remove biofilms and necrotic tissues while leaving healthy tissue undamaged. Wound necrotic tissue and biofilms are stained so that they can be photographed by a capture system for image analysis. The unique approach of the proposed system uses a narrow (2mm x 2mm) plasma scalpel that can be moved by a robotic arm to a desired location over a wound in response to real time imaging of the stained wound. A computer algorithm processes the images to determine the location of the stained material in the wound. A control system moves the scalpel to the desired location and delivers a stream of plasma to selectively remove only necrotic tissue/biofilms and not healthy tissue. The rate of etching would be dynamically controlled by altering plasma energy and rate of passage over the afflicted area. The objectives of this proposal are to (1) demonstrate that a CAP plasma scalpel system can image and remove biofilms from solid substrates and (2) show selective removal of biofilms from model tissues (Matrigel) and in ex vivo porcine ear models that more closely resemble the complex cellular environments of true wounds. To accomplish these objectives, a workforce of predominantly undergraduates will conduct experiments to examine the plasma ablation of biofilms grown on glass coverslips and other substrates (Matrigel, etc) using staining and microscopic imaging, followed by an analysis of surviving colony forming units (cfu) to demonstrate microbial cell killing. Profilometry across the treated biofilm will be used to demonstrate the depth and width of sample removal from the site including over-etch of healthy tissue. The results will be used in iterative rounds of scalpel design to improve the performance of the instrument. Subsequent experiments will demonstrate selectivity by using the device to treat model ex vivo porcine ear wounds that contain biofilms in the context of viable mammalian tissue. Wound bioburden will be analyzed through a combination of staining/imaging techniques. Ablation of wound bioburden will be examined by cfu reduction assays using standard plating techniques of wound rinses, histological sampling, staining and imaging using standard light microscopy. Selectivity will be further demonstrated using live-dead staining and fluorescence confocal microscopy to show that the underlying healthy tissue is unaffected by plasma treatment.