Anisotropic DBSCAN for 3D SMLM Data Clustering

Single-molecule localization microscopy (SMLM) advanced biological discoveries beyond the diffraction limit. Various implementations enable 3D SMLM to reconstruct volumetric cell images. Yet, the inherent anisotropic point spread function of optical microscopes often limits the localization precision in the axial direction compared to the lateral precision. Such localization anisotropy could also expand spherical cellular structures to ellipsoidal cellular structures. Structure identification, however, is often performed using DBSCAN cluster algorithms, considering an isotropic search volume. Here, we show that an anisotropic DBSCAN search volume identifies anisotropic clusters more reliably using simulated ground truth data sets. Given experimental localization precisions, we suggest optimized search parameters based on an expanded computational grid search and show an enhanced performance of anisotropic DBSCAN amidst variations in localization precision. We demonstrate the capability of anisotropic DBSCAN on experimental data and anticipate that the algorithm allows for a more rigorous identification of clusters in cells, considering the anisotropic localization precisions of astigmatism-based 3D SMLM.