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- Fig. 1.
Imaging the nuclear envelope by SEM and dSTORM. (A) SEM imaging is restricted to the nucleoplasmatic, nuclear-basket-containing, side, whereas dSTORM imaging is performed from the cytoplasmatic side, where the anchoring proteins are oriented in the direction of the coverglass. Schematically, an electron beam and an objective are depicted to symbolize SEM and dSTORM imaging, respectively. (B) SEM image of a folded nuclear envelope. (C) Influence of the pixel binning used to reconstruct the super-resolved dSTORM image on resolution, that is, the visualization of the central channel of the NPC. Scale bar: 200 nm.
- Fig. 2.
Correlative dSTORM-SEM of NPCs. (A) dSTORM image of the central channel labeled with WGA–Alexa-647, (B) corresponding SEM image of the nucleoplasmatic side of the nuclear envelope and (C) overlay of the dSTORM and SEM images. (D) Detailed overlay of a different area, highlighting that some NPCs are not labeled by WGA–Alexa-647. (E) The integral membrane protein gp210 was labeled using immunofluorescence with Alexa 647. (F) Corresponding SEM image, (G) overlay of dSTORM and SEM image and (H) overlay of the detailed eightfold structure of the outer gp210 ring. (I,J) Examples of NPCs revealing a ninefold symmetrical structure of gp210 proteins (Hinshaw and Milligan, 2003). Scale bars: 250 nm (A–C), 100 nm (D), 500 nm (E–G), 200 nm (H–J).
- Fig. 3.
Localization statistic of gp210 proteins labeled with a primary antibody and a secondary Alexa-647-labeled F(ab′)2 fragment. The mean localization number was determined as 274±4 for whole NPCs (an example is shown in the inset) (A), 35.1±0.5 for single domains (dashed ring in inset) within the eightfold NPC structure (B), and 8.1±0.2 for isolated fluorescence signals (spots, dashed ring in inset) found in NPCs corresponding to an individual F(ab′)2 fragment (C) (mean±s.e.m.). Scale bars, 20 nm.
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