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. Author manuscript; available in PMC: 2020 Apr 7.
Published in final edited form as: Methods. 2018 Aug 18;153:46–62. doi: 10.1016/j.ymeth.2018.08.005

Fig. 2.

Fig. 2.

Determination of the central axis of the NPC illuminated by SPEED microscopy. Diagrams are used to demonstrate the detailed steps in our data analyses. (A) The initial single-molecule tracking data (black points) are plotted around the NPC’s marker (red dot) collected with SPEED. (B) The collected data after filtering by using single-molecule spatial localization precision. (C) The filtered 2D single-molecule data shape the spatial location of the NE and a single NPC. The red dot represents the location of the NPC’s marker. But sometimes the marker’s position is not perfectly overlapped with the averaged central positions in either x or y dimension suggested by the 2D single-molecule data. Also, the 2D single-molecule distribution also indicates the orientation of NPC. If the orientation of the NPC is within a free angle of 1.4° to the perpendicular direction to the NE [51], the 2D single-molecule data will be proceeded further. If it is bigger, the data will be dropped. (D) The plot of projected locations of these 2D single-molecule data in the y dimension will indicate if a fine adjustment for the central cytoplasmic transport axis is needed or not. Here we just show an example that peak 1 (p1) and peak 2 (p2) are not symmetrical locating at −23 nm and 21 nm respectively. If the peaks are symmetrical, the step E will be skipped to move onto the 2D to 3D transformation directly. (E) The two peaks are averaged as (p1+p22), resulting a fine adjustment in the NPC’s central cytoplasmic transport axis. The dotted red line has shifted to the corrected central position. (F and G) Similar to the y-dimensional data process, we next determine the precise location of the NPC’s central position along the x dimension by fitting the histogram of these 2D single-molecule locations within the NPC’s scaffold region projected into the x dimension (ranging from −20 nm to 20 nm, as clearly shown in the void region of the NE in (C) even if the NPC’s marker is a little bit off sometimes). (H) The 2D single-molecule data with the confirmed x and y axes will undergo the 2D to 3D transformation (Fig. 3) to produce the 3D density map of mRNP’s nuclear export routes (red clouds).