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Title
Japanese: 
English:Single Molecule Imaging and Nanomanipulation of Biomolecules 
Author
Japanese: Yoshie Harada, Takashi Funatsu, 徳永 万喜洋, Kiwamu Saito, Hideo Higuchi, Yoshiharu Ishii, Toshio Yanagida.  
English: Yoshie Harada, Takashi Funatsu, Makio Tokunaga, Kiwamu Saito, Hideo Higuchi, Yoshiharu Ishii, Toshio Yanagida.  
Language English 
Journal/Book name
Japanese: 
English:METHODS IN CELL BIOLOGY 
Volume, Number, Page Vol. 55        pp. 117-128
Published date 1998 
Publisher
Japanese: 
English: 
Conference name
Japanese: 
English: 
Conference site
Japanese: 
English: 
Abstract The best way to provide unambiguous information about the working principle of biomolecular machines such as molecular motors is to directly observe and manipulate individual molecular machines under an optical microscope. Because of the diffraction limit, however, the resolution of conventional optical microscopes(～0.2 μm)is too low for observing single protein molecules, or even their small assembly. Thus, a great deal of effort has been made to increase the resolution of the experimental system. One method is to observe the scattered light from objects by using a dark-field microscope equipped with a very strong illuminating source and a highly sensitive camera. However, the objects observed need to be as large as a microtubule (～20 nm) in diameter (Hotani, 1976). A similar resolution can be achieved if the contrast of the recording is greatly enhanced by computer image processing (video-enhanced contrast method)(Inoue, 1981). Fluorescence microscopy is useful for observing smaller objects. About 10 yeart ago, it was demonstrated that single actin filaments (double-helical polymer of actin), labeled with fluorescent phalloidin, can be clearly seen by fluorescence microscopy (Yanagida et al., 1984). This finding led to the development of new in vitro motility assays that address the elementary process of force generation by actin-myosin interaction directly at the molecular level(Harada et al, 1987; Kishino and Yanagida, 1988; Kron and Spudich, 1986; Toyoshima et al., 1987). More recently combining of techniques for manipulation of an actin filament and nanometry has allowed the individual mechanical events driven by adenosine 5'-triphosphate (ATP) hydrolysis to be measured at sub-piconewton and sub-nanometer resolutions directly from multiple (Finer et al., 1994) and single myosin molecules (Ishijima et al., 1991; Ishijima et al., 1994). The number of photons emitted from a single fluorophore excited by a strong light source such as a laser is sufficient for it to be visualized by a commercial high-sensitivity video camera. Thus, it is theoretically possible to visualize single biomolecules labeled with a fluorescent dye, but until recently it was not yet proven in proctice owing to great background noise. Recently, new fluorescence microscopes have been developed with 20- to more than 2000-fold less background noise than a conventional fluorescence microscope and thus enable visualizing single fluorophores in aqueous solution (Fanatsu et al., 1995). Here, we demonstrate that the movements and individual ATP turnovers of single motor proteins can be directly visualized at the real time.

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