Two-photon microscopy is a less-invasive cross-sectional imaging technique for long-term visualization of living cells within deeper layers of organs. This microscopy is based on the multi-photon excitation process and has been used widely in medical and biological sciences. An attractive property of two-photon microscopy, multicolor excitation capability has enabled quantification of spatiotemporal patterns of [Ca(2+)]i, ion transport and single episodes of fusion pore openings during exocytosis. In pancreatic acinar cells, we have successfully demonstrated the existence of "sequential compound exocyotosis" for the first time. Sequential compound exocytosis has subsequently been identified in a wide variety of secretory cells including exocrine, endocrine and blood cells. Further exploration has revealed dynamics and physiological roles of actin cytoskeleton, and soluble NSF attachment receptor (SNARE) proteins. In addition, our newly developed method (TEPIQ method) can be used to determine fusion pores and the diameters of vesicles smaller than the diffraction-limited resolution. Recently, we have successfully observed neurons deeper than 0.9 mm from the brain cortex surface in an anesthetized mouse. We have also improved the spatial resolution needed to visualize fine structures of basal dendrites in layer V in vivo. This microscopy also can be used to visualize dendritic spines, axon terminals and miroglia cells, suggesting that we can follow long-term changes of neural or glial cells in a living mouse. Two-photon microscopy will thus be important in advancing the study of the molecular basis of physiological and pathological events in the human body.