Project Summary Catheter ablation (CA) is a potentially curative treatment for nearly all cardiac arrhythmias, yet clinical outcomes remain suboptimal, leading to repeat procedures. Success rates would improve if all targets for ablation could be identified and if real-time assessment of the durability and continuity of ablation lesions could be determined. These fundamental gaps in knowledge could be overcome by applying spectroscopic photoacoustic imaging (sPAI) techniques to intraoperatively guide and monitor CA. sPAI can provide insight into tissue characterization and ablation lesions based on wavelength-dependent optical absorption differences of tissue. By targeting hemoglobin (total, deoxy-, and oxy-hemoglobin) and water absorption differences, perfused/viable tissue along with local tissue oxygen saturation (StO2) and water content can be determined. The depth of this imaging can go beyond the endocardial or epicardial surfaces of the heart and provide novel tissue characterization in the “mid-myocardial” region of the heart, which is presently unknown via current mapping techniques. Prior work by our group has shown that sPAI can provide real-time quantification of thermal ablation extent and depth. These data are currently unavailable with existing technologies used by clinicians. Consequently, the goal of our research is to develop and validate real-time sPAI techniques assessing tissue oxygen saturation (StO2), total hemoglobin, and water content to 1) identify and differentiate regions of viable myocardium versus scar and 2) determine permanently ablated tissue versus temporarily “injured” yet still viable myocardium. Such a capability promises to identify novel targets for ablation, which would directly improve CA outcomes. Current approaches often cannot directly identify deeper “mid-myocardial” targets for CA and adjunctive ablation techniques to target these regions are based either on operator experience and/or prior failed procedures. sPAI would have the ability to create—for the first time—a standardized workflow for when and how to target currently difficult to access regions of the myocardium. This would have profound clinical implications for CA success rates while reducing procedural complications. We intend to accomplish these aims by first optimizing sPAI techniques with ultrasound (US) incorporation in ex-vivo ventricular tissue. This will determine the specifications needed for optimal (e.g., maximal depth penetration and StO2 accuracy) tissue imaging. Subsequent work will then be focused on optimization of in-vivo, sPAI-based, myocardial tissue characterization using an open-chest porcine infarct model. We intend to identify regions of viable myocardium and scar and validate with grossly co-registered MRI and independent histopathologic assessment. Finally, building on these initial experiments, we intend to differentiate permanently ablated tissue from surrounding edematous (“injured”) and normal myocardial tissue i...