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

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 APD₇₀ and APD₈₀, with bias confidence intervals including zero. Cumulative probability analysis revealed a marked improvement in precision following combined motion correction, with the proportion of optical action potential duration (APD) measurements within ±10 ms of MAP increasing from 16.9%, 29.2% and 26.2% in raw recordings to 75.4%, 87.7% and 80% for APD₅₀, APD₇₀ and APD₈₀, respectively. Collectively, these findings establish the integrated motion-correction strategy as a validated framework for accurate quantification of action potentials in contracting hearts, supporting more physiologically relevant optical mapping. NEW & NOTEWORTHY: While motion tracking and ratiometric approaches have previously been used to mitigate motion artefacts in optical mapping, this study is the first to integrate emission ratiometry with fully marker-free motion tracking and to provide systematic validation against simultaneously recorded monophasic action potentials in freely contracting isolated rabbit hearts. This validated framework enables accurate optical assessment of cardiac electrical activity under physiologically relevant conditions without mechanical stabilization or excitation-contraction uncoupling.