Project Summary: Atrial fibrillation (AF) is the most frequent cardiac arrhythmia, and it is a major risk factor for ischemic stroke and provokes morbidity and mortality along with a significant economic burden. Although AF has been studied in various animals, the embryonic zebrafish has been the genetically tractable and optically transparent model to investigate electromechanical coupling during cardiac development. Like in humans, the action potential and the consequent myocardial contraction are also key indicators of cardiac function in the zebrafish. By virtual of its transparency, optical mapping has been a primary means to investigate the interplay between cardiac action potential and myocardial contraction to study the mechanisms of AF. Dysregulation of electrical and mechanical coupling is a significant factor underlying the pathogenesis and perpetuation of AF. Optical mapping of electromechanical decoupling in zebrafish is nontrivial because it requires simultaneous recording of fast propagating voltage waves and myocardial contraction. Particularly in a beating heart, the rapid myocardial contraction can easily blur the image—the motion artifacts superimpose the wave patterns appearing in the optical maps and can prohibit further analysis of the imaging data. Pharmacological uncoupling has been widely used to suppress heart motion. However, this makes studying electromechanical coupling impossible. Alternatively, post-acquisition synchronization approach records a z-stack of movies, each covering at least one cardiac cycle. After the recording is completed, one 3D cardiac cycle can be reconstructed by synchronizing the movies in time. Nonetheless, this method is inapplicable to nonperiodic movements, such as irregular heartbeats with AF. Therefore, there is an unmet need to develop innovative optical techniques for high-speed 3D mapping of electromechanical coupling in a rapidly and irregularly beating AF heart. To solve this problem, we propose to develop a light-sheet light-field tomography (light-sheet LIFT) technique for kilohertz 3D imaging of electromechanical coupling in zebrafish hearts undergoing AF. Our method has only recently become possible due to two emerging technologies, light-field tomography (LIFT) and light-sheet microscopy, both of which we have extensive experience with. We will integrate LIFT with light-sheet microscopy and enable high-resolution 3D imaging with an unprecedented volumetric frame rate. The resultant system, light- sheet LIFT, will provide enough spatiotemporal resolution to fully depict the interplay between voltage waves, myocardial contraction, and intracardiac blood flow in a pitx2c zebrafish arrhythmia model. We expect our method will advance the understanding of AF's fundamental mechanism from the electrical activities at a single- cell level.