To elucidate the spatiotemporal profiles of final secretory stage, we have established two-photon extracellular polar tracer (TEP) imaging, with which we can quantify all exocytic events in the plane of focus within the intact tissues. With such technique, we can estimate the precise diameters of vesicles independently of the spatial resolution of optical microscope, and measure the fusion pore dynamics at nanometer resolution. At insulin exocytosis in the pancreatic islets, it took two seconds for the fusion pore to dilate from 1.4 nm in diameter to 6 nm in diameter, and such unusual stability of the pore may be due to the crystallization of the intragranular contents. Opening of the pore was preceded by unrestricted lateral diffusion of lipids along the inner wall of the pores, supporting the idea that this structure was mainly composed of membrane lipids. TEP imaging has been also applied to other representative secretory glands, and has revealed hitherto unexpected diversity in spatial organizations of exocytosis and endocytosis, which are relevant for physiology and pathology of secretory tissues. In the pancreatic islet, compound exocytosis was characteristically inhibited (<5%), partly due to the rarity of SNAP25 redistribution into the exocytosed vesicle membrane. Such mechanisms necessitate transport of insulin granules to the cell surface for fusion, and possibly rendering exocytosis more sensitive to metabolic state. Two-photon imaging will be powerful tools to elucidate molecular and cellular mechanisms of exocytosis and related disease, and to develop new therapeutic agencies as well as diagnostic tools.