Two-dimensional imaging of fast intracellular Ca2+ release

Asynchronous release of calcium (Ca(2+))-for example, the generation of Ca(2+) alternans in cardiac myocytes-is a phenomenon important in the development of cardiac arrhythmogenesis. The development of a failure to release Ca(2+) at individual release sites can be regarded as a major contributor to cardiac pathologies such as hypertrophy. Although confocal linescans provide sufficient temporal resolution to investigate the physiological and pathological cardiac excitation-contraction (EC) coupling, linescans can only image ∼1.5% of the cross section of myocytes, which raises doubts about how representative such recordings are, especially in light of nonhomogeneous uncoupling of Ca(2+) channels and ryanodine receptors. Nowadays, the speed of confocal microscopes has been greatly improved, enabling two-dimensional (2D) imaging at sufficient image rates (>100 frames/sec). To understand better the physiological and pathophysiological EC coupling of cardiomyocytes, we describe here a protocol to monitor fast intracellular Ca(2+) signals using fast 2D confocal scanning.