High-Speed Angle-Resolved Imaging of a Single Gold
Nanorod with Microsecond Temporal Resolution and One-Degree Angle
Precision
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Abstract
We
developed two types of high-speed angle-resolved imaging methods
for single gold nanorods (SAuNRs) using objective-type vertical illumination
dark-field microscopy and a high-speed CMOS camera to achieve microsecond
temporal and one-degree angle resolution. These methods are based
on: (i) an intensity analysis of focused images of SAuNR split into
two orthogonally polarized components and (ii) the analysis of defocused
SAuNR images. We determined the angle precision (statistical error)
and accuracy (systematic error) of the resultant SAuNR (80 nm ×
40 nm) images projected onto a substrate surface (azimuthal angle)
in both methods. Although both methods showed a similar precision
of ∼1° for the azimuthal angle at a 10 μs temporal
resolution, the defocused image analysis showed a superior angle accuracy
of ∼5°. In addition, the polar angle was also determined
from the defocused SAuNR images with a precision of ∼1°,
by fitting with simulated images. By taking advantage of the defocused
image method’s full revolution measurement range in the azimuthal
angle, the rotation of the rotary molecular motor, F<sub>1</sub>-ATPase,
was measured with 3.3 μs temporal resolution. The time constants
of the pauses waiting for the elementary steps of the ATP hydrolysis
reaction and the torque generated in the mechanical steps have been
successfully estimated. The high-speed angle-resolved SAuNR imaging
methods will be applicable to the monitoring of the fast conformational
changes of many biological molecular machines