SuperResNET GUI: 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). The computational modules of SuperResNET include correction for multiple blinking of a single fluorophore, denoising, segmentation (clustering), and feature extraction, which are then used for cluster group identification, modularity analysis, blob retrieval and visualization in 2D and 3D. Using publicly available dSTORM data, we apply a graphical user interface (GUI) version of SuperResNET to nucleoporin Nup96 structures, that present a highly organized octagon structure comprised of eight corners. SuperResNET GUI effectively segments nuclear pores and Nup96 corners based on differential proximity threshold analysis. SuperResNET GUI quantitatively analyzes features from segmented nuclear pore structures, including complete structures with 8-fold symmetry, and from segmented corners. SuperResNET GUI modularity analysis of segmented corners distinguishes two modules at 11.1 nm distance, corresponding to two individual Nup96 molecules. SuperResNET GUI 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 GUI provides flexibility to report on structural diversity in situ within the cell without model-fitting, providing opportunities for biological discovery.