Abstract
We report an automated single particle tracking technique for tracking the x, y, z coordinates, azimuthal and elevation angles of anisotropic plasmonic gold nanorod probes in live cells. These five spatial coordinates are collectively referred to as 5D. This method overcomes a long-standing challenge in distinguishing rotational motions from translational motions in the z-axis in differential interference contrast microscopy to result in full disclosure of nanoscale motions with high accuracy. Transferrin-coated endocytic gold nanorod cargoes initially undergo active rotational diffusion and display characteristic rotational motions on the membrane. Then as the cargoes being enclosed in clathrin-coated pits, they slow down the active rotation and experience a quiet period before they restore active rotational diffusion after fission and eventually being transported away from the original entry spots. Finally, the 3D trajectories and the accompanying rotational motions of the cargoes are resolved accurately to render the intracellular transport process in live cells.
Highlights
We report an automated single particle tracking technique for tracking the x, y, z coordinates, azimuthal and elevation angles of anisotropic plasmonic gold nanorod probes in live cells
The single particle orientation and rotational tracking (SPORT) technique previously developed in our laboratory takes the advantage of the plasmon resonance-associated birefringence of gold nanorods[25, 27, 29, 34] and Nomarski-type differential interference contrast (DIC) microscopy[35, 36], which utilizes two orthogonally polarized beams for illumination
The new 5D-Single particle tracking (SPT) technique uses parallax to sense the axial movement of the target object, and at the same time, employs an automatic feedback loop algorithm to control the focal plane of the objective and bring the target object back to focus repetitively
Summary
We report an automated single particle tracking technique for tracking the x, y, z coordinates, azimuthal and elevation angles of anisotropic plasmonic gold nanorod probes in live cells. These five spatial coordinates are collectively referred to as 5D. It is difficult to resolve the dipole orientation of single fluorescent probes in a cellular environment with reasonably fast temporal resolution due to the well-known limitations, such as high background and fast photobleaching This challenge can be circumvented by using anisotropic plasmonic gold nanorods as alternative rotational tracking probes. This ambiguity would result in incorrect assignment of the dipole’s orientation
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