Combined emission ratiometry and motion tracking for optical mapping of contracting hearts: Validation with monophasic action potentials.

Cardiac optical mapping in fully contracting hearts is limited by motion artefacts, leading most investigations to rely on excitation-contraction uncoupling despite its impact on physiological relevance. Although motion tracking and ratiometric imaging have each been used to reduce motion artefacts, and their combined use has been demonstrated with excitation ratiometry, systematic validation of emission ratiometry combined with motion tracking against an established electrophysiological reference remains limited. In this study, we integrated emission ratiometry with two-dimensional, marker-free motion tracking to suppress motion-related artefacts during optical mapping of Langendorff-perfused rabbit hearts, leveraging the advantage that emission ratiometry derives both signals from the same excitation pathway and thereby reduces sensitivity to motion-related photometric fluctuations in fluorescence intensity. Motion-corrected optical action potentials were quantitatively validated against simultaneously recorded monophasic action potentials (MAPs), with optical signals extracted from regions adjacent to the MAP electrode. Compared with raw recordings and with either method applied alone, the combined approach produced the closest agreement with MAP-derived repolarization measurements. Bland-Altman analysis indicated no systematic bias at APD