Multidimensional detection and analysis of Ca2+ sparks in cardiac myocytes

Examining calcium spark morphology and its relationship to the structure of the cardiac myocyte offers a direct means of understanding excitation-contraction coupling mechanisms. Traditional confocal line scanning achieves excellent temporal spark resolution but at the cost of spatial information in the perpendicular dimension. To address this, we developed a methodology to identify and analyze sparks obtained via two-dimensional confocal or charge-coupled device microscopy. The technique consists of nonlinearly subtracting the background fluorescence, thresholding the data on the basis of noise level, and then localizing the spark peaks via a generalized extrema test, while taking care to detect and separate adjacent peaks. In this article, we describe the algorithm, compare its performance to a previously validated spark detection algorithm, and demonstrate it by applying it to both a synthetic replica and an experimental preparation of a two-dimensional isotropic myocyte monolayer exhibiting sparks during a calcium transient. We find that our multidimensional algorithm provides better sensitivity than the conventional method under conditions of temporally heterogeneous background fluorescence, and the inclusion of peak segmentation reduces false negative rates when spark density is high. Our algorithm is robust and can be effectively used with different imaging modalities and allows spark identification and quantification in subcellular, cellular, and tissue preparations.