Extended Data Fig. 3: Tracking the motion of nanorotor AuNPs and determination of the angular position.
From: The energy landscape for R-loop formation by the CRISPR–Cas Cascade complex
a, Stack of consecutive images of an individual 50 nm AuNP attached to a DNA nanorotor construct recorded at 3947 fps. The image size is 31 px × 31 px corresponding to a field of view of 1.28 µm × 1.28 µm. b, Localization of the AuNP. To determine the position of the AuNP centre, the intensity distribution of a 13 px × 13 px area (pink square) around the pixel with maximum intensity was fitted with a two-dimensional Gaussian function (pink circles, indicating Gaussian intensities of 6000, 5000, 4000, 3000, 2000, 1000 from inside to outside). Measurements on AuNPs stuck to the glass substrate yielded a resolution of 2.7 nm. c, Corrections for magnetic bead fluctuations and drift of the microscope. In parallel to the tracking of the AuNP, we record the positions of the attached magnetic bead and a surface-attached non-magnetic reference bead. The position tracking of the reference bead is used to remove the thermal drift of the microscope. Additionally, thermal fluctuations cause lateral displacements x of the magnetic bead from the equilibrium position. In turn this results in lateral displacements xb of the nanorotor rotation centre. To correct for this, we determined xb from xb=h⁄L⋅x, where L is the distance of the magnetic bead centre from the DNA surface attachment (obtained from bead tracking) and h is the height of the AuNP above the surface for which 118 nm was taken. d, Tracked positions of a nanorotor bound AuNP in the XY plane. Imperfections of the magnet configuration can cause a tilt of the pulling direction with respect to the optical axis, providing an elliptic distortion of the AuNP positions due to the tilt of the rotation plane. A fit of the data with an ellipse is shown in orange. To correct for the tilt of the rotational plane, the short ellipse axis was stretched to the value of the long axis to yield a circular trajectory (shown in red). This correction was applied to all data points in order to obtain correct angular positions of the nanorotor.
