High-speed angle-resolved imaging of a single gold nanorod with microsecond temporal resolution and one-degree angle precision.

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 x 40 nm) images projected onto a substrate surface (azimuthal angle) in both methods. Although both methods showed a similar precision of similar to 1 degrees for the azimuthal angle at a 10 mu s temporal resolution, the defocused image analysis showed a superior angle accuracy of similar to 5 degrees. In addition, the polar angle was also determined from the defocused SAuNR images with a precision of similar to 1 degrees, by fitting with simulated images. By taking advantage of the defocused image methods full revolution measurement range in the azimuthal angle, the rotation of the rotary molecular motor, F-1-ATPase, was measured with 3.3 mu s 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.