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

通过设计一个光学与电流扫描仪，提供了一个荧光照明的能力，以更深的一部分的细胞比消逝显微镜，我们观察到3种不同类型的细胞的分泌囊泡，特别注意的囊泡的运动前胞吐。渐逝光照明的穿透深度是从由于盖片与标准盖片重叠而引起的亮点的移位来测量的。在肾上腺素分泌细胞中，细胞外pH的变化不影响胞吐的闪光模式，表明囊泡内pH的升高不是闪光反应的原因。在表达GFP-胰岛素的INS-1细胞中，在胞吐闪光反应之前，一些囊泡迅速移位到细胞膜。我们发现这种移位发生在细胞膜的深度变化。在这些细胞中，很少看到易位后立即胞吐。在MIN-6细胞中，许多小泡平行于细胞膜长距离滑动。这些囊泡很少发生胞吐闪光反应。从这些观察，我们得出结论，闪光反应表明货物材料从囊泡的扩散。进一步发展能够在短时间内测量物体速度的显微光学是必要的，我们目前正沿着这条路线工作。

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.