The development of technologies that allow for live optical imaging of exocytosis from β-cells has greatly improved our understanding of insulin secretion. Two-photon imaging, in particular, has enabled researchers to visualize the exocytosis of large dense-core vesicles (LDCVs) containing insulin from β-cells in intact islets of Langerhans. These studies have revealed that high glucose levels induce two phases of insulin secretion and that this release is dependent upon cytosolic Ca2+ and cAMP. This technology has also made it possible to examine the spatial profile of insulin exocytosis in these tissues and compare that profile with those of other secretory glands. Such studies have led to the discovery of the massive exocytosis of synaptic-like microvesicles (SLMVs) in β-cells. These imaging studies have also helped clarify facets of insulin exocytosis that cannot be properly addressed using the currently available electrophysiological techniques. This chapter provides a concise introduction to the field of optical imaging for those researchers who wish to characterize exocytosis from β-cells in the islets of Langerhans.