Real-time imaging of synaptic vesicle exocytosis by total internal reflection fluorescence (TIRF) microscopy.

Synaptic vesicles are one of the smallest organelle in the cell with their sizes far below the diffraction limit of the light microscopy. Exocytosis at the synapse is tightly regulated reaction which typically occurs within a millisecond after the arrival of an action potential. It has been assumed that synaptic vesicles have to be ready for immediate exocytosis upon the arrival of final trigger before exocytosis. But direct observation of the pre-exocytotic synaptic vesicle dynamics have been lacking. Total internal reflection microscopy (TIRFM) is a fluorescence microscopy which has best z-axis resolution (∼100 nm) as a light microscopy, and is close to that of the ultrathin section used for electron microscopy. Although its application is limited to the objects just beneath the plasma membrane, TIRFM has revealed dynamics of various organelles and proteins. We recently managed to dissociate mammalian neuronal presynaptic terminals and let the exocytotic sites adhere tightly to the coverslip. There, TIRFM revealed the detailed dynamics of pre-exocytotic vesicles. Our work opened up the way to visualize dynamics of sub-diffraction limited sized organelle in a real time, and will be useful for direct visualization of various synaptic components in the future.