Numerous signaling processes in the cell are controlled in microdomains that are defined by cellular structures ranging from nm to μm in size. Recent improvements in microscopy enable the resolution and reconstruction of these micro domains, while new computational methods provide the means to elucidate their functional roles. Collectively these tools allow for a biophysical understanding of the cellular environment and its pathological progression in disease. Here we review recent advancements in microscopy, and subcellular modeling on the basis of reconstructed geometries, with a special focus on signaling microdomains that are important for the excitation contraction coupling in cardiac myocytes.