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。为了测试这一点，我使用了瞬变显微镜和带有微透镜的尼普科夫圆盘扫描仪的共聚焦显微镜。通过这些技术，从嗜铬囊泡释放的儿茶酚胺和在非洲爪哇卵母细胞中表达的K-cahnnels的门控活性被捕捉为动态二维图像，首先将荧光染料Quacine加载到嗜铬囊泡中，然后用配备大数值孔径物镜的共聚焦显微镜观察染料的荧光，从而可视化儿茶酚胺的胞吐释放。研究发现，一些囊泡只部分释放其内容物，并在没有完全释放的情况下循环使用。这一发现并不支持被广泛接受的跨安非他明的量子释放假说。K通道的信使RNA在351位被修饰，因此在注入该信使RNA的Xenpus卵母细胞中表达的K通道可以被四甲基罗丹明马来酰亚胺(TMRM)特异性标记。在电压钳条件下，用荧光显微镜观察TMRM在卵母细胞膜上的荧光。许多荧光点的亮度随电压而变化(幅度为50%，去极化为100 mV)，反映了离子通道的选通过程。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，就像预期的单个荧光分子一样。这是首次在活体细胞质膜上观察到单分子的证据。

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.