title: Simple dark-field microscopy with nanometer spatial precision and microsecond temporal resolution.
abstract: Molecular motors such as kinesin, myosin, and F(1)-ATPase are responsible for many important cellular processes. These motor proteins exhibit nanometer-scale, stepwise movements on micro- to millisecond timescales. So far, methods developed to measure these small and fast movements with high spatial and temporal resolution require relatively complicated experimental systems. Here, we describe a simple dark-field imaging system that employs objective-type evanescent illumination to selectively illuminate a thin layer on the coverslip and thus yield images with high signal/noise ratios. Only by substituting the dichroic mirror in conventional objective-type total internal reflection fluorescence microscope with a perforated mirror, were nanometer spatial precision and microsecond temporal resolution simultaneously achieved. This system was applied to the study of the rotary mechanism of F(1)-ATPase. The fluctuation of a gold nanoparticle attached to the gamma-subunit during catalytic dwell and the stepping motion during torque generation were successfully visualized with 9.1-mus temporal resolution. Because of the simple optics, this system will be applicable to various biophysical studies requiring high spatial and temporal resolution in vitro and also in vivo.
authors.affiliation: Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueno
authors.forename: So
authors.initials: S
authors.lastname: Nishikawa
authors.forename: Ryota
authors.initials: R
authors.lastname: Iino
authors.forename: Kazuhito V
authors.initials: KV
authors.lastname: Tabata
authors.forename: Shouichi
authors.initials: S
authors.lastname: Sakakihara
authors.forename: Toshio
authors.initials: T
authors.lastname: Yanagida
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Noji
mesh_terms: D001407:Bacillus / Q000201:enzymology; D003624:Darkness; D005898:Glass / Q000737:chemistry; D006046:Gold / Q000737:chemistry; D008029:Lighting; D053768:Metal Nanoparticles / Q000737:chemistry; D008853:Microscopy / Q000295:instrumentation / Q000379:methods*; D057054:Molecular Imaging / Q000295:instrumentation / Q000379:methods*; D009068:Movement; D036103:Nanotechnology; D006180:Proton-Translocating ATPases / Q000378:metabolism; D013997:Time Factors; D014867:Water / Q000737:chemistry
chemical_list: D014867:Water; D006046:Gold; D006180:Proton-Translocating ATPases