Fig. 4
Fixed and live 2D localization of nucleoporins-POM121 labeled with blinking fluorophores. Fixed and time-lapse images of the reconstructed three-dimensional distribution of nucleoporin molecules. a 3D maximum intensity projections for NPCs structures of a fixed and intact HeLa cell expressing POM121-Halo and labeled with Halo-HMSiR dyes. The localized sites are displayed as a white point cloud while DAPI fluorescence nuclear staining is colored in blue. Scale bar is 3 µm. b The high magnification area of the white box in (a) with one NPC structure marked in red. After fitting by a donut function using least-square method, the top 50 optimal fits (shown as the montage in (c)) were centered and overlaid together as (d) with scale bar 100 nm. e The 2D reconstructed image on a live cell with POM121 labeled with HMSiR reconstructed based on 10,000 frames. The local density was plotted with temporal color code. f A magnified area in (e) is enlarged to show the temporal evolution of the distribution of NPC. Two selected cases are plotted with moving directions in the insets. g Images reconstructed from certain time interval are serially plotted to reveal change of the spatial distribution with respect to time (the contrast of (f, g) are adjusted to enhance the clustered feature of NPC)
