Quantification of molecular dynamics, numbers and interactions of multi-kind molecules by single molecule imaging in cells

我们已经开发出技术，可以在单分子水平上可视化细胞功能，如免疫反应和信号传递过程。单分子成像与同时显示细胞中几种不同蛋白质的能力相结合，使分子动力学、相互作用和动力学的量化成为可能。这些三维和时间参数使我们能够对细胞功能进行数值模拟和计算机模拟，为细胞作为分子系统的功能开辟新的前沿。我们已经证明，细胞中单个分子的清晰可视化使其分子定量成为可能。利用TIRF和HELO显微镜实现了清晰的单分子可视化。单分子荧光成像的主要技术挑战是提高信号/背景比。我们参与了全内反射荧光显微镜(TIRF)的开发，这是一种光镜TE…更多的创意是使用逝去的光来照亮单分子。TIRF已经成为一种广泛用于表面单分子成像的技术，但由于消失光的深度非常有限，无法用于单细胞成像。我们通过倾斜照明光束和最小化照明面积，在克服这一限制方面取得了显着的成功。高倾斜和层叠光学薄片(HILO)显微镜是建立在TIRF方法的基础上的。由高度倾斜的细光束照明增加了图像强度，降低了背景强度，产生的信号/背景比大约是表观照明的8倍。最重要的是，HELO不仅可以用来显示细胞表面的单分子，也可以用来显示活细胞内的单分子。使用HALO和TIRF的单分子显微镜可以用于对光照敏感的活细胞内的观察，以及由于只需要微弱的光照而进行长时间的延时观察。利用HELO，可以追踪细胞内大量单分子的动态运动。因此，HILO对于定量研究大量分子的分布、动态运动或相互作用也是有用的。我们已经安装了一个新的多色系统来更详细地观察分子间的相互作用。高S/N比的图像也可以产生清晰的三维图像。我们从核孔复合体的系列图像中重建了三维图像。这表明，高光显微镜使研究人员能够可视化三维分子图像，而传统的共聚焦显微镜无法重建这些图像，因为荧光染料在三维扫描过程中会发生光漂白。较少

We have developed technologies that allow cellular functions such as immunological responses and signaling processes are to be visualized at the single-molecule level. Single molecule imaging coupled with the ability to simultaneously visualize several different proteins in cells has enabled the quantification of molecular dynamics, interactions, and kinetics. These three-dimensional and temporal parameters enables us to carry out numerical modeling and computer simulations of cell functions, to open up new frontiers for cellular functions as molecular systems.We have demonstrated that clear visualization of single molecules in cells enablesd their molecular quantification. Clear single-molecule visualization was achieved using TIRF and HILO microscopy. The main technical challenge of single-molecule fluorescence imaging is increasing the signal/background ratio. We have been involved in the development of total internal reflection fluorescence (TIRF) microscopy, a light-microscopic te … More chnique that uses evanescent light to illuminate single molecules. TIRF has become a widespread technique for single-molecule imaging at surfaces, but cannot be used for single-cell imaging due to the very limited depth of evanescent light. We have achieved notable success in overcoming this limitation by inclining the illumination beam and by minimizing the illumination area.Highly inclined and laminated optical sheet (HILO) microscopy is built upon the TIRF approach. Illumination by a highly inclined and thin beam increases image intensity and decreases background intensity, yielding a signal/background ratio about eightfold greater than that of epi-illumination. Most importantly, HILO can be used to visualize single molecules not only on cell surfaces but also inside living cells.Single molecule microscopy using HILO and TIRF can be used for observations within living cells that are sensitive to the illuminated light, and for time-lapse observation over long periods since only weak illumination is required. Using HILO, the dynamic movements of large numbers of single molecules inside a cell can be traced. Thus, HILO is also useful for quantitative studies of the distribution, dynamic movement, or interaction of large numbers of molecules. We have installed a new multi-color system to observe intermolecular interactions in ever greater detail.A high S/N ratio of images by HILO microscopy also yields clear three-dimensional images. We reconstructed a three-dimensional image from serial images of nuclear pore complexes. It demonstrated that HILO microscopy enables researchers to visualize three-dimensional molecular images which cannot be reconstructed by conventional confocal microscopy because of photo-bleaching of fluorescent dyes during three-dimensional scanning. Less