Visulization of a single molecule in a living cell

这个项目的目的是可视化活细胞中分子的实时图像。为了验证这一点，我使用了消失显微镜和带有微型透镜的尼普科夫圆盘扫描仪的共聚焦显微镜。利用这些技术，以动态二维图像的形式捕获了爪蟾卵母细胞中染色质囊泡中儿茶酚胺的释放和k -通道的门控活性。儿茶酚胺的胞外释放是通过将荧光染料醌（quinacrine）加载到染色质囊泡中，然后用配备高数值孔径物镜的共聚焦显微镜观察染料的荧光来观察的。研究发现，有些囊泡仅部分释放其内容物，并在不完全释放的情况下再循环。这一发现不支持被广泛接受的发射机量子释放假说。将K-channel的信使RNA在351位点进行修饰，使其能够被四甲基罗丹明马来酰亚胺（TMRM）特异性标记在Xenpus卵母细胞中表达的K-channel。在电压箝位条件下，用消失显微镜观察TMRM在卵母细胞膜上的荧光。许多荧光点显示出电压依赖性的亮度变化（振幅为50%，退极化为100mV），这反映了离子通道的门控过程。此外，随着光照时间的延长，这些斑点以量子漂白的方式突然消失，正如对单个荧光分子的预期那样。这是首次在活细胞的质膜上观察到单个分子的证据。

The purpose of this project is to visualize real-time images of molecules in living cells. To test this, I employed the evanescence microscopy and the confocal microscopy with a microlens-attached Nipkow disk scanner. By these techniques, catecholamine release from chromaffin vesicles and gating activity of K-cahnnels expressed in Xenopus oocytes were captured as dynamic 2-dimesnional images.Exocytotic release of catecholamine was visualized by first loading a fluorescent dye, quinacrine, into chromaffin vesicles, and then by observing the fluorescence of the dye with the confocal microscope equipped with an objective lens of a high numerical aperture. It was found that some vesicles release their contents only partially and recycle without a full release. This findings do not support the widely accepted hypothesis of quantal release of tranamitters.Messenger RNA for K-channel was modified at 351 position, so that K-channel expressed in Xenpus oocytes injected with the mRNA can be specifically labeled with tetramthyl rhodamine maleimide (TMRM). Under the voltage clamp condition, the fluorescence of TMRM on the oocyte membrane was examined with the evanescence microscope. Many fluorescent spots showed voltage dependent changes in brightness (50% in amplitude with 100mV depolarization) that reflects the gating process of the ion channel. Moreover, with the time of illumination these spots disappeared abruptly in a mode of quantal bleaching, as expected for a single fluorescent molecule. This is evidence for the first observation of single molecules on the plasma membrane in a livinf cell.