Cardiac optical mapping uses potentiometric fluorescent dyes to image membrane potential (V-m). An important limitation of conventional optical mapping is that contraction is usually arrested pharmacologically to prevent motion artifacts from obscuring V-m signals. However, these agents may alter electrophysiology, and by abolishing contraction, also prevent optical mapping from being used to study coupling between electrical and mechanical function. Here, we present a method to simultaneously map V-m and epicardial contraction in the beating heart. Isolated perfused swine hearts were stained with di-4-ANEPPS and fiducial markers were glued to the epicardium for motion tracking. The heart was imaged at 750 Hz with a video camera. Fluorescence was excited with cyan or blue LEDs on alternating camera frames, thus providing a 375-Hz effective sampling rate. Marker tracking enabled the pixel(s) imaging any epicardial site within the marked region to be identified in each camera frame. Cyan-and blue-elicited fluorescence have different sensitivities to V-m, but other signal features, primarily motion artifacts, are common. Thus, taking the ratio of fluorescence emitted by a motion-tracked epicardial site in adjacent frames removes artifacts, leaving V-m (excitation ratiometry). Reconstructed V-m signals were validated by comparison to monophasic action potentials and to conventional optical mapping signals. Binocular imaging with additional video cameras enabled marker motion to be tracked in three dimensions. From these data, epicardial deformation during the cardiac cycle was quantified by computing finite strain fields. We show that the method can simultaneously map V-m and strain in a left-sided working heart preparation and can image changes in both electrical and mechanical function 5 min after the induction of regional ischemia. By allowing high-resolution optical mapping in the absence of electromechanical uncoupling agents, the method relieves a long-standing limitation of optical mapping and has potential to enhance new studies in coupled cardiac electromechanics.
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