The evanescence microscope with a high resolving power is a useful tool for the observation of molecular dynamics. However, an accurate tracking of molecules in the intracellular space is difficult because the evanescent wave for the fluorescence excitation is restricted only to the area close to the coverslip. To overcome this problem and to study the dynamic events in the cytoplasm, we modified the evanescence microscope with an ultrahigh NA objective lens. We employed a galvanomirror to aim and switch the laser beam rapidly at the back focal plane of an 1.65 NA objective near its periphery so that the penetration depth of evanescent wave varied depending on the beam-incidence angles at the interface. Under this microscope, trafficking of secretory vesicles was studied in MIN-6 cells expressing insulin-GFP. Stimulation of the cell with glucose induced a transient increase in GFP fluorescence, but evanescence images acquired alternately at a 0.5 s interval were slightly different in spite of the same field of view. This result reflects temporal and spatial dynamics of secretory vesicles prior to exocytosis. The ultrahigh NA lens provides a large window for evanescent wave illumination with a wide range of penetration depth, thus is useful for analyzing 3D events in the cell. [J Physiol Sci. 2006;56 Suppl:S247]

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