Validation of the resolution limit of 3D-ExM using microtubules and NPCs. (A) Representative images of microtubules with or without using different ExM methods (pre-ExM, 4×, and 12× 3D-ExM) and microscopes (conventional confocal and STED). (B) Consecutive serial sections (400 nm for each step) of antibody-stained microtubules in PtK2 cells after 12× 3D-ExM. (C) Representative images of RPE1 cells stained with anti-Nup107 antibody and DNA dye before expansion. (D) Representative 12× 3D-ExM images of RPE1 cells stained with anti-Elys antibody. Blue arrows indicate NPs at the edge of the nucleus. The three images within the orange bounding box are zoomed-in images of the indicated area in the left image. (E) Quantification of the frequency of NPs possessing Nup107 dots. n = 751 NPs pooled from six cells from two biological replicates. (F) Quantification of the measured and corrected surface area of RPE1 nuclei in 12× 3D-ExM images. n = 15 cells pooled from three biological replicates. DNA was stained with DAPI (pre-ExM) or DRAQ5 (post-ExM).