Development of Evanescence Microscope and Test of the Quantal Hypothesis for Exocytosis

为了研制一种基于超高NA透镜的高性能隐失显微镜，比较了三种引入照明光的方法。光纤照明方法在安全性、视野大小和易于操作方面优于直接照明方法，但成本高且稍不稳定。用渐逝光显微镜观察嗜铬细胞和β细胞的胞吐作用。在这两种类型的细胞中，荧光探针从具有闪光响应的囊泡中释放。闪光反应的幅度是可变的，这取决于囊泡。它反映了水从囊泡分泌。通过激光捕获力谱，我们证明了细胞在刺激时对水施加的小力。囊泡的内容物不是通过简单的扩散而是通过囊泡中的水喷射而释放。喷射力的强度随囊泡的不同而变化，因为通过免疫染色技术检查的囊泡膜上的Cl通道的密度不同。Cl通道阻断剂抑制闪光反应，但不抑制胞吐作用。在β细胞中，囊泡在其胞吐反应后立即沿膜沿着移动，表明囊泡内容物的持续释放。这些发现与所谓的量子假说不相容。在内分泌细胞中，分泌功能的调节是以高度复杂的方式进化的，以便能够对内部和外部环境进行良好的适应。

Aiming at development of a user-friendly high-performance evanescence microscope based on an ultra-high NA lens, we compared three methods of introducing the illumination light to the microscope. A fiber-illumination method was advantageous in safety, field size, and easy handling, over a direct-illumination method, but was costly and slightly unstable. Using an evanescence microscope, we examined the exocytosis in chromaffin cells and beta cells. In both types of the cell, a fluorescent probe was released from vesicles with a flash response. The flash response was variable in amplitude depending on vesicles. It reflected water secretion from the vesicles. By laser-trap forcemetry, we demonstrated a small force exerted on water by the cell upon stimulation. The contents of vesicles are released not by a simply diffusion but by a water ejection from the vesicles. The strength of the ejecting force varied with vesicles as the density of Cl channels on the vesicle membrane examined by an immuno-staining technique varied. Cl channel blockers suppressed the flash response without suppressing the exocytosis. In beta cells, vesicle moved along the membrane immediately after its exocytotic response, suggesting a continuous release of vesicle contents. These findings are incompatible with the so-called quantal hypothesis. In endocrine cells, modulation for the secretory function is evolved in a highly complex manner so that fine adaptations to inner and outer environments are made possible.