Steps to determine the diameter of the nuclear pore membrane. (A) Nuclear pore predictions for all the pores on the nuclear envelope of naïve interphase cell 19 (Hela-2) prepared by HPFS and visualized at 4 × 4 × 5.3 nm isotropic resolution. The nuclear pore predictions were obtained using a model trained without fine tuning with ground truth annotations for Cell 13 (Hela) prepared by HPFS and imaged at ∼5 nm isotropic resolution. (B) Volume location of the centroid of each of the predicted nuclear pore, color coded according to their relative position along the Z-axis (top panel) and surface rendition of the nuclear envelope (green) obtain by alpha-shape triangulation of the centroids (see Materials and methods). Orthonormal vectors associated with each triangle are shown (red). (C) Example of realignment of a nuclear pore from its acquisition orientation in the FIB-SEM volume image to a new view with the nuclear envelope orthogonal to the Z-axis; side views and volume rendition of the nuclear pore prediction are shown. (D) Single plane on the face and orthogonal views of a nuclear pore centered on the middle of the nuclear envelope (left panels) and examples of the intensity plots used to estimate the membrane pore diameters by determining the distance separating the two intensity minima along the indicated axis (right panels). The nuclear pore diameter is reported as the average of 18 values obtained 10° apart (inset in left panel).