Development of Fluorescence Microscopy for Visualizing Single Biomolecules in Living Cells

利用尼普科夫圆盘共聚焦显微镜首次对水溶液中的单个荧光分子进行视频成像。用四甲基罗丹明（TMR）或IC5荧光染料标记的单个激酶分子成像来评价该方法的性能。在入射激光功率为0.5 W/mm^2的情况下，TMR和IC5荧光团的光分解寿命分别为~ 10秒和~ 2秒。荧光强度和光漂白率与激光功率成正比，范围在0.65 ~ 3 W/mm^2之间。利用该显微镜可以观察到单个驱动蛋白分子沿玻璃表面微管的二维滑动运动，以及单个驱动蛋白分子在粘性溶液中的三维布朗运动。这些结果表明，该方法可用于活细胞中单分子事件的实时研究。

Single fluorescent molecules in aqueous solution were imaged for the first time at video-rate using Nipkow disk-type confocal microscopy. Performance of this method was evaluated by imaging single kinesin molecules labeled with fluorescent dyes of tetramethylrhodamine (TMR) or IC5. Photodecomposition lifetimes of the fluorophores were 〜10 sec for TMR and 〜2 sec for IC5 under the incident laser power of 0.5 W/mm^2. Both the fluorescence intensity and the photobleaching rate were proportional to the laser power from 0.65 to 3 W/mm^2. 2D sliding movement of single kinesin molecules along microtubules on glass surface, and 3D Brownian motion of individual kinesin molecules in viscous solution could be observed using this microscopy. These results indicated that this method could be applicable to the study of single molecular events in living cells at real time.