Gold Nanorod Single-Molecule Rotation Assay
AuNR and Light Scattering
Gold nanorods (AuNR) scatter red polarized light from the long axis, and green polarized light from the short axis.
Control
Attach a AuNR directly to a glass surface so that it is unable to rotate. Illuminate it and monitor the scattered red light intensity from it with a single photon counter. When you periodically rotate the polarizer, the intensity of scattered red light changes in a sinusoidal manner. The error in rotary position of the AuNR relative to the polarizer is then determined from the distribution in intensities at each rotary position. It is small.
Now attach a AuNR to the rotor of an F₁ motor, add ATP so the motor rotates, and repeat the experiment above. If you monitor light intensity at ~1000 frames per min, the F₁ rotary power strokes are too fast to capture the intermediate positions. So you mostly see when the motor is dwelling (pausing) between power strokes. Since F₁ dwells every 120°, you see 3 sinusoidal curves, one for each dwell. So you know it is rotating.
Computing Rotational Position
Using dark field microscopy, a field of view of AuNR attached to F₁-ATPase molecules that are actively rotating blink red and green when viewed through the polarizer at 50 frames per sec.
Note that in this field of view ~80% are blinking. We typically see >50%, while most other assays observe a much smaller percentage of actively rotating molecules.
We then move the stage to align one rotating molecule with a pinhole that allows the light from only that one AuNR. The red light then goes through a polarizer and is collected by the single photon counter.
The polarizer is rotated on the chosen F₁ molecule so that the scattered light intensity from the AuNR is at a minimum at one of the catalytic dwells.
Light intensity vs. time is recorded at 400 kHz (400,000 frames per sec, i.e. every 2.5 μs).
As the AuNR rotates during the subsequent power stroke, intensity will increase through a maximum at 90°, then decrease until it stops rotating at 120° (red dots).
Rotational position vs. time are calculated using an arcsine-square-root function for thousands of these power strokes.
Determining Rotational Direction
Rotational direction is determined by using a beam splitter to divert half of the scattered light intensity to a second detector and off-set polarizers.
Rotate the polarizer of detector-2 relative to detector-1 so that maximum scattered light intensity occurs later for detector-2 when the AuNR is rotating counter clockwise, and occurs earlier when rotating clockwise.