DISTINCTION OF THE MECHANISMS FOR EXOCYTOSIS BY SIMULTANEOUS MEASUREMENTS WITH EVANESCENCE METHOD AND CONFOCAL METHOD

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By devising an optics with the galvano-scanner that provides a capability of fluorescence illumination to a deeper part of cells than the evanescence microscopy, we observed secretory vesicles of 3 different types of cells with a special attention to the movement of the vesicles before exocytosis. The penetration depths of the evanescence illuminations were measured from the shift of the bright spot due to overlaying of a coverslip to the standard coverslip. In adrenaline secreting cells, a change in extracellular pH was not affecting to the flash pattern of the exocytosis, indicating that the rise in pH inside vesicles is not cause for the flash response. In INS-1 cells, expressing GFP-insulin, some vesicles translocated rapidly to the cell membrane before exocytotic flash response. We found such a translocation occurred toward the cell membrane as depth change. In these cells, it was rare to see translocations immediately followed by exocytosis. In MIN-6 cells, many vesicles glided for long distances in parallel to the cell membrane. These vesicles rarely underwent the exocytotic flash response. From these observations, we concluded that the flash response indicates the diffusion of cargo materials from vesicles. A further development of microscopic optics that can measure the velocity of object in short time would be necessary, and we are currently working along this line.