Figure 2. 3D passive diffusion routes of differently charged GFPs.

(A) −30 GFP. Calculated electrostatic surface potentials of GFP variants, ranging from −15 kT/e (dark blue) to +15 kT/e (dark red); neutral charge 0 is shown in green (I). Superimposed plots of thousands of localizations of single molecules located primarily within a rectangular area of 240×160 nm (II). N, the nucleoplasmic side of the NPC. C, the cytoplasmic side of the NPC. The locations in each 10×10 nm area were quantized and filtered with a Gaussian blur function to generate the 2D probability density map overlaid onto the NPC architecture (light blue). The highest density was 1×10⁵ locations/µm², and the lowest was 0 locations/µm², shown in gray (III). A 3D probability density map generated using 2D to 3D deconvolution algorithms (red cloud; brighter color indicates higher density) is shown in both side-view and a top-view orientations superimposed on the NPC architecture (blue). The length of the path and the diameter at the central plane of the NPC were measured and marked in nanometers (IV). N, the nucleoplasmic side of the NPC. C, the cytoplasmic side of the NPC. (B–C) Nucleocytoplasmic transport pathways of −7 GFP and +36 GFP.