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

通过设计一种光学与电流扫描仪，提供荧光照明的细胞较深的部分比消失显微镜的能力，我们观察了3种不同类型的细胞分泌囊泡，特别注意囊泡的运动前的胞外分泌。消光照明的穿透深度由覆盖盖盖到标准盖盖的亮点位移来测量。在肾上腺素分泌细胞中，细胞外pH值的变化对胞吐的闪光模式没有影响，表明囊泡内pH值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，一些囊泡在胞吐闪光反应之前迅速转移到细胞膜上。我们发现，随着深度的变化，这种移位发生在细胞膜上。在这些细胞中，很少看到易位后立即出现胞吐。在MIN-6细胞中，许多囊泡沿着平行于细胞膜的方向滑行很长一段距离。这些囊泡很少发生胞外闪反应。从这些观察中，我们得出结论，闪光反应表明货物物质从囊泡扩散。进一步发展可以在短时间内测量物体速度的微观光学是必要的，我们目前正在沿着这条路线工作。

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.