Simple dark-field microscopy with nanometer spatial precision and microsecond temporal resolution.
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.
Ueno
Hiroshi
H
Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan.
Nishikawa
So
S
Iino
Ryota
R
Tabata
Kazuhito V
KV
Sakakihara
Shouichi
S
Yanagida
Toshio
T
Noji
Hiroyuki
H
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
D014867:Water; D006046:Gold; D006180:Proton-Translocating ATPases