Figure 1. Transport of single molecules through a single NPC tracked by SPEED microscopy and further characterized with 2D to 3D deconvolution algorithms.

(A) Diagram of the illumination of a single GFP-NPC (blue) and the tracking of single fluorescent in-transit molecules (red dots) through this NPC in cells by SPEED microscopy. SPEED microscopy generates an inclined illumination point spread function (iPSF forms an angle of 45° to the z direction) in the focal plane (between the double light blue lines) at the equatorial plane of a cell nucleus. C, cytoplasm. N, nucleus. (B) A well-isolated fluorescent spot of a single GFP-NPC (green) overlaid with a single in-transit fluorescent molecule (SM, red spot, here using Alexa Fluor 647 labeled single GFP molecule as an example) captured by SPEED microscopy. A typical nuclear transport event of a single molecule (red spot) from the nucleus to the cytoplasm is captured at 0.4 ms per frame. Numbers denote time in milliseconds. Scale bar, 1 µm. (C) Using a 2D Gaussian fit of isolated fluorescent spots, the trajectories of an in-transit molecule (red dots) and the centroid of the NPC (the blue dot) were obtained and then overlaid onto the NPC architecture (blue). Clearly, the transiting molecule interacted with the NPC from 0.8 ms to 2.8 ms. N, the nucleoplasmic side of the NPC. C, the cytoplasmic side of the NPC. (D) The diagram illustrates the 2D to 3D deconvolution process. In principle, 2D spatial locations (x, y) projected from 3D locations (x, y, z) in a Cartesian coordinate system retain all of the information about the 3D locations that can also be encoded in a cylindrical coordinate system (R, θ, x). Following an established approach, here a deconvolution process (or a back-projection process) was set up between a simplified cylindrical coordinate system (R, constant, x) and the obtained 2D spatial locations (x, y). Detailed deconvolution process can be found in previous publications [27], [30].