BackgroundHeterogeneities of repolarization (R) across the myocardium have been invoked to explain most reentrant arrhythmias. The measurement of refractory periods (RPs) has been widely used to assess R, but conventional electrode and extrastimulus mapping techniques have not provided reliable maps of RPs.Methods and ResultsGuinea pig hearts were stained with a voltage-sensitive dye to measure fluorescence (F) action potentials (APs) from 124 sites with a photodiode array. AP duration (APD) was defined as the time between depolarization (dF/dt)maxand R time points (ie, the time when AP returns to baseline or some percent thereof). However, R time points are difficult to determine because AP downstrokes are often encumbered by drifting baselines and motion artifacts, which make this definition ambiguous. In optical and microelectrode recordings, the second derivative of AP downstrokes is shown to contain an easily detected, unique local maximum. The correlation between the position of this maximum (d2F/dt2)max, and R has been tested during altered AP characteristics induced by changes in cycle length, ischemia, and hypoxia. Under these various modifications of the AP, the time points of (d2F/dt2)maxfell at 97.0±2.1% of recovery to baseline. Extrastimulus techniques applied to (1) isolated myocytes, (2) intact hearts, and (3) mathematical simulations indicated that (d2V/dt2)maxcoincided with the effective RPs of APs. The coincidence of RPs and (d2V/dt2)maxwas valid within 5 milliseconds, for resting potentials of −75 to −90 mV and extrastimuli three times threshold voltage.ConclusionsThus, optical APs and (d2F/dt2)maxcan be used to map activation, R, and RPs with AP recordings from a single heartbeat.