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

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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