Conceptual Considerations

Excitation–contraction coupling is an essential mechanism responsible for the blood-pumping function of the heart. In cardiac myocytes, repetitive Ca2+ cycling translates electric excitation (the action potential) into force generation (contrac-tion).1 Altered Ca2+ handling has been related to many cardiac diseases, such as cardiac hypertrophy2,3 and heart failure.4,5 Therefore, the elucidation of the initiation and progression of cardiac diseases via monitoring Ca2+ handling, either at the cellular level or in tissue, is of great interest. Fluorescence-based intracellular Ca2+ imaging is the most versatile method for monitoring Ca2+ concentrations in living cells or tissues.6–8 Fluorescence-based Ca2+ imaging typically uses Ca2+-sensitive fluorescent dyes, such as small organic molecules and, to a lesser extent, genetically encoded Ca2+ in-dicators (GECIs). GECIs represent a new generation of Ca2+