Ca²⁺ sparks within cardiomyocytes are a result of local sarcoplasmic reticulum (SR) Ca²⁺ release due to the transient opening of a cluster of ryanodine receptors (RyRs). Typically the characteristics of Ca²⁺ sparks within these cells are obtained by analysis of line-scan images generated by laser scanning confocal microscopy (LSCM). This is performed using epi-fluorescence excitation of the Ca²⁺ sensitive dye Fluo-3. The detection algorithm (Cheng et al. 1999) depends on identifying Ca²⁺ spark events as transient fluorescent signals that are greater than the mean fluorescence value plus an arbitrary amplitude value (CRI) times the standard deviation of the signal. One method, to measure the optimum CRI value is to record the signal from cells in the absence of RyR activity (using SR inhibitors). The CRI value is then set to minimise the number of false events (Lukyanenko et al. 2000). However, this approach will exclude small amplitude Ca²⁺ spark events from the analysis.

We have developed a new method that allows for detection of actual events and estimation of false positives in the same cell. This approach involves performing line-scan recordings before and immediately after the inhibition of RyR activity. Application of the detection algorithm after inhibition of RyR yields an amplitude distribution histogram that reflects purely false event detection. This profile can be subtracted from the initial recording to yield a histogram that represents the contribution of events due to RyR activity (Fig. 1). The permeabilised cardiomyocyte lends itself to this approach since intracellular [Ca²⁺] can be maintained before and after the application of SR inhibitors.