SuperResNET: Model‐Free Single‐Molecule Network Analysis Software Achieves Molecular Resolution of Nup96

SuperResNET is an integrated machine learning-based analysis software for visualizing and quantifying 3D point cloud data acquired by single-molecule localization microscopy (SMLM). SuperResNET computational modules include correction for multiple blinking of single fluorophores, denoising, segmentation (clustering), feature extraction used for cluster group identification, modularity analysis, blob retrieval, and visualization in 2D and 3D. Here, a graphical user interface version of SuperResNET was applied to publicly available direct stochastic optical reconstruction microscopy (dSTORM) data of nucleoporin Nup96 and Nup107 labeled nuclear pores that present a highly organized octagon structure of eight corners. SuperResNET effectively segments nuclear pores and Nup96 corners based on differential proximity threshold analysis from 2D and 3D SMLM datasets. SuperResNET quantitatively analyzes features from segmented nuclear pores, including complete structures with eightfold symmetry, and from segmented corners. SuperResNET modularity analysis of segmented corners from 2D SMLM distinguishes two modules at 10.7 ± 0.1 nm distance, corresponding to two individual Nup96 molecules. SuperResNET is therefore a model-free tool that can reconstruct network architecture and molecular distribution of subcellular structures without the bias of a specified prior model, attaining molecular resolution from dSTORM data. SuperResNET provides flexibility to report on structural diversity in situ within the cell, providing opportunities for biological discovery.