Fig. 4.

MINFIELD STED nanoscopy of samples with low fluorophore-to-protein ratio. Here, Abberior Star Red-labeled nanobodies are used to visualize the arrangement of Nup93 and Nup98 within the amphibian NPC. Nup93 forms a ring around the center of the nuclear pore with a diameter of ∼70 nm. Its substructure (A) would be obscured by the size of the antibody tree in conventional indirect immunolabeling. Using nanobodies reduces the number of dye molecules per protein considerably. (B) Limiting the scan field compensates this effect and images detailed cellular structures with high SNR at ∼20-nm resolution. C shows a line profile through the Nup93 image at the indicated position. The data were fitted with two Gaussians, yielding a peak-to-peak separation smaller than 23 nm and a full width at half-maximum of ∼15 nm for the individual Gaussians. (Scale bars: 20 nm.)