AbstractClustering of proteins is crucial for many cellular processes and can be imaged at nanoscale resolution using single-molecule localization microscopy (SMLM). Ideally, molecular clustering in regions of interest (ROIs) from SMLM images would be assessed using computational methods that are robust to sample and experimental heterogeneity, account for uncertainties in localization data, can analyze both 2D and 3D data, and have practical computational requirements in terms of time and hardware. While analyzing surface protein clustering on B lymphocytes using SMLM, we encountered limitations with existing cluster analysis methods. This inspired us to develop StormGraph, an algorithm using graph theory and community detection to identify clusters in heterogeneous sets of 2D and 3D SMLM data while accounting for localization uncertainties. StormGraph generates both multi-level and single-level clusterings and can quantify cluster overlap for two-color SMLM data. Importantly, StormGraph automatically determines scale-dependent thresholds from the data using scale-independent input parameters. This makes identical choices of input parameter values suitable for disparate ROIs, eliminating the need to tune parameters for different ROIs in heterogeneous SMLM datasets. We show that StormGraph outperforms existing algorithms at analyzing heterogeneous sets of simulated SMLM ROIs where ground-truth clusters are known. Applying StormGraph to real SMLM data in 2D, we reveal that B-cell antigen receptors (BCRs) reside in a heterogeneous combination of small and large clusters following stimulation, which suggests for the first time that two conflicting models of BCR activation are not mutually exclusive. We also demonstrate application of StormGraph to real two-color and 3D SMLM data.
Artifact Formation in Single Molecule Localization Microscopy
