Fig 4. Quantitative localization microscopy with heterogeneous labeling density.

(a) Overview image (pixel size 10 nm, clipped for visibility) and (b) zoomed inset (pixel size 4 nm) of the dashed white box in (a) of secondary antibody-Alexa Fluor 647 labeled Nup153 protein of the NPC in the nuclear membrane with non-specifically bound (secondary) antibodies outside the nuclear membrane region. (c) The correlation parameter Q for the region inside the nuclear membrane (red box) is higher than outside (blue box) due to the tight clustering of the secondary antibodies labeling the Nup153 proteins. The relative number of accumulated localizations at each time point is similar, indicating that the bleaching behavior is similar and the sources of the localizations are identical in both regions.