The islet of Langerhans is the functional unit responsible for glucose-modulated insulin and glucagon secretion, and thus plays a key role in blood glucose homeostasis. We are interested in understanding the molecular mechanisms of islet function, and their role in the regulation of blood glucose under normal and pathological conditions. Using quantitative optical imaging of metabolism, membrane potential, free Ca2+, and enzymatic activation, the dynamics of these mechanisms can be measured in islets and even in living animals. Glucose-stimulated insulin secretion is controlled by the activity of glucokinase (GK), and we have shown that GK is regulated by association with other cellular constituents. To determine the preferred interaction partners for GK, we have utilized Förster resonance energy transfer (FRET), which is widely used to study biomolecular dynamics and protein interactions in live cells. Many issues complicate FRET measurements. We have developed two novel approaches approach for absolute and high precision measurements of FRET efficiency, one based on lock-in detection of an optical switch acceptor and a second based on snapshot hyperspectral imaging. These approaches will be described and their use for measuring intracellular protein interactions will be discussed. Downstream of GK signaling, we have shown that gap junction coupling between islet cells regulates their membrane polarization, although other coupling mechanisms also play a role. We have introduced precise experimental perturbations in both the gap junction coupling and the individual cell membrane potentials. We find that decreasing gap junction coupling can lead to sub-regions becoming electrically active, but that these active sub-regions do not lead to increased insulin secretion. We have optimized mathematical models of coupled β-cell electrophysiology to describe accurately these variations in electrical activity as a function of coupling.