Detailed understanding of the adaptive nature of cardiac cells in health and disease requires investigation of proteins and membranes in their native physiological environment, ideally by noninvasive optical methods. However, conventional light microscopy does not resolve the spatial characteristics of small fluorescently labeled protein or membrane structures in cells. Due to diffraction limiting resolution to half the wavelength of light, adjacent fluorescent molecules spaced at less than ~ 250 nm are not separately visualized. This fundamental problem has lead to a rapidly growing area of research, superresolution fluorescence microscopy, also called nanoscopy. We discuss pioneering applications of superresolution microscopy relevant to the heart, emphasizing different nanoscopy strategies toward new insight in cardiac cell biology. Here, we focus on molecular and structural readouts from subcellular nanodomains and organelles related to Ca2 + signaling during excitation–contraction (EC) coupling, including live cell imaging strategies. Based on existing data and superresolution techniques, we suggest that an important future aim will be subcellular in situ structure–function analysis with nanometric resolving power in organotypic cells. This article is part of a Special Issue entitled “Calcium Signaling in Heart”.