{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,27]],"date-time":"2026-02-27T06:13:17Z","timestamp":1772172797387,"version":"3.50.1"},"update-to":[{"DOI":"10.1371\/journal.pcbi.1008479","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2020,12,18]],"date-time":"2020-12-18T00:00:00Z","timestamp":1608249600000}}],"reference-count":28,"publisher":"Public Library of Science (PLoS)","issue":"12","license":[{"start":{"date-parts":[[2020,12,8]],"date-time":"2020-12-08T00:00:00Z","timestamp":1607385600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000038","name":"Natural Sciences and Engineering Research Council of Canada","doi-asserted-by":"publisher","award":["RGPIN-2019-06520"],"award-info":[{"award-number":["RGPIN-2019-06520"]}],"id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000038","name":"Natural Sciences and Engineering Research Council of Canada","doi-asserted-by":"publisher","award":["RGPIN-2019-06520"],"award-info":[{"award-number":["RGPIN-2019-06520"]}],"id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000038","name":"Natural Sciences and Engineering Research Council of Canada","doi-asserted-by":"crossref","award":["RGPIN-2019-06520"],"award-info":[{"award-number":["RGPIN-2019-06520"]}],"id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003400","name":"Ontario Ministry of Research, Innovation and Science","doi-asserted-by":"publisher","award":["ER14-10-182"],"award-info":[{"award-number":["ER14-10-182"]}],"id":[{"id":"10.13039\/501100003400","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003400","name":"Ontario Ministry of Research, Innovation and Science","doi-asserted-by":"publisher","award":["ER14-10-182"],"award-info":[{"award-number":["ER14-10-182"]}],"id":[{"id":"10.13039\/501100003400","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.ploscompbiol.org"],"crossmark-restriction":false},"short-container-title":["PLoS Comput Biol"],"abstract":"<jats:p>Single-molecule localization microscopy (SMLM) is a powerful tool for studying intracellular structure and macromolecular organization at the nanoscale. The increasingly massive pointillistic data sets generated by SMLM require the development of new and highly efficient quantification tools. Here we present FOCAL3D, an accurate, flexible and exceedingly fast (scaling linearly with the number of localizations) density-based algorithm for quantifying spatial clustering in large 3D SMLM data sets. Unlike DBSCAN, which is perhaps the most commonly employed density-based clustering algorithm, an optimum set of parameters for FOCAL3D may be objectively determined. We initially validate the performance of FOCAL3D on simulated datasets at varying noise levels and for a range of cluster sizes. These simulated datasets are used to illustrate the parametric insensitivity of the algorithm, in contrast to DBSCAN, and clustering metrics such as the F1 and Silhouette score indicate that FOCAL3D is highly accurate, even in the presence of significant background noise and mixed populations of variable sized clusters, once optimized. We then apply FOCAL3D to 3D astigmatic dSTORM images of the nuclear pore complex (NPC) in human osteosaracoma cells, illustrating both the validity of the parameter optimization and the ability of the algorithm to accurately cluster complex, heterogeneous 3D clusters in a biological dataset. FOCAL3D is provided as an open source software package written in Python.<\/jats:p>","DOI":"10.1371\/journal.pcbi.1008479","type":"journal-article","created":{"date-parts":[[2020,12,8]],"date-time":"2020-12-08T16:27:00Z","timestamp":1607444820000},"page":"e1008479","update-policy":"https:\/\/doi.org\/10.1371\/journal.pcbi.corrections_policy","source":"Crossref","is-referenced-by-count":8,"title":["FOCAL3D: A 3-dimensional clustering package for single-molecule localization microscopy"],"prefix":"10.1371","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3913-4810","authenticated-orcid":true,"given":"Daniel F.","family":"Nino","sequence":"first","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2996-8547","authenticated-orcid":true,"given":"Daniel","family":"Djayakarsana","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2515-4025","authenticated-orcid":true,"given":"Joshua N.","family":"Milstein","sequence":"additional","affiliation":[]}],"member":"340","published-online":{"date-parts":[[2020,12,8]]},"reference":[{"issue":"36","key":"pcbi.1008479.ref001","doi-asserted-by":"crossref","first-page":"14919","DOI":"10.1039\/c3cp52289j","article-title":"Single-molecule localization microscopy-near-molecular spatial resolution in light microscopy with photoswitchable fluorophores","volume":"15","author":"A F\u00fcrstenberg","year":"2013","journal-title":"Phys Chem Chem Phys"},{"issue":"5793","key":"pcbi.1008479.ref002","doi-asserted-by":"crossref","first-page":"1642","DOI":"10.1126\/science.1127344","article-title":"Imaging intracellular fluorescent proteins at nanometer resolution","volume":"313","author":"E Betzig","year":"2006","journal-title":"Science (New York, NY)"},{"issue":"7","key":"pcbi.1008479.ref003","doi-asserted-by":"crossref","first-page":"991","DOI":"10.1038\/nprot.2011.336","article-title":"Direct stochastic optical reconstruction microscopy with standard fluorescent probes","volume":"6","author":"S van de Linde","year":"2011","journal-title":"Nature protocols"},{"issue":"11","key":"pcbi.1008479.ref004","doi-asserted-by":"crossref","first-page":"4756","DOI":"10.1021\/nl103427w","article-title":"Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami","volume":"10","author":"R Jungmann","year":"2010","journal-title":"Nano Lett"},{"issue":"7","key":"pcbi.1008479.ref005","doi-asserted-by":"crossref","first-page":"1047","DOI":"10.1016\/j.cell.2010.12.002","article-title":"Breaking the diffraction barrier: super-resolution imaging of cells","volume":"143","author":"B Huang","year":"2010","journal-title":"Cell"},{"issue":"10","key":"pcbi.1008479.ref006","doi-asserted-by":"crossref","first-page":"1607","DOI":"10.1242\/jcs.080085","article-title":"Super-resolution microscopy at a glance","volume":"124","author":"CG Galbraith","year":"2011","journal-title":"Journal of Cell Science"},{"issue":"7306","key":"pcbi.1008479.ref007","doi-asserted-by":"crossref","first-page":"647","DOI":"10.1038\/nature09163","article-title":"Subnanometre single-molecule localization, registration and distance measurements","volume":"466","author":"A Pertsinidis","year":"2010","journal-title":"Nature"},{"issue":"6325","key":"pcbi.1008479.ref008","doi-asserted-by":"crossref","first-page":"606","DOI":"10.1126\/science.aak9913","article-title":"Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes","volume":"355","author":"F Balzarotti","year":"2017","journal-title":"Science"},{"issue":"2","key":"pcbi.1008479.ref009","doi-asserted-by":"crossref","first-page":"345","DOI":"10.1038\/nprot.2013.005","article-title":"Quantifying spatial organization in point-localization superresolution images using pair correlation analysis","volume":"8","author":"P Sengupta","year":"2013","journal-title":"Nature Protocols"},{"issue":"7","key":"pcbi.1008479.ref010","doi-asserted-by":"crossref","first-page":"446","DOI":"10.1002\/jbio.200900089","article-title":"PALM imaging and cluster analysis of protein heterogeneity at the cell surface","volume":"3","author":"DM Owen","year":"2010","journal-title":"Journal of Biophotonics"},{"key":"pcbi.1008479.ref011","unstructured":"Ester M, Kriegel HP, Sander J, Xu X. A Density-based Algorithm for Discovering Clusters a Density-based Algorithm for Discovering Clusters in Large Spatial Databases with Noise. In: Proceedings of the Second International Conference on Knowledge Discovery and Data Mining. KDD\u201996. AAAI Press; 1996. p. 226\u2013231."},{"issue":"3","key":"pcbi.1008479.ref012","doi-asserted-by":"crossref","first-page":"100038","DOI":"10.1016\/j.patter.2020.100038","article-title":"A Review of Super-Resolution Single-Molecule Localization Microscopy Cluster Analysis and Quantification Methods","volume":"1","author":"IM Khater","year":"2020","journal-title":"Patterns"},{"issue":"11","key":"pcbi.1008479.ref013","doi-asserted-by":"crossref","first-page":"837","DOI":"10.1016\/j.tcb.2020.07.005","article-title":"Quantitative Data Analysis in Single-Molecule Localization Microscopy","volume":"30","author":"YL Wu","year":"2020","journal-title":"Trends Cell Biol"},{"issue":"5","key":"pcbi.1008479.ref014","doi-asserted-by":"crossref","first-page":"747","DOI":"10.1093\/bioinformatics\/btv630","article-title":"Fast Optimized Cluster Algorithm for Localizations (FOCAL): a spatial cluster analysis for super-resolved microscopy","volume":"32","author":"A 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