{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T14:54:59Z","timestamp":1785855299519,"version":"3.56.0"},"reference-count":16,"publisher":"Wiley","issue":"7","license":[{"start":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T00:00:00Z","timestamp":1783987200000},"content-version":"vor","delay-in-days":13,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T00:00:00Z","timestamp":1782864000000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["1R15GM135806\u201001"],"award-info":[{"award-number":["1R15GM135806\u201001"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["2R15GM135806\u201001"],"award-info":[{"award-number":["2R15GM135806\u201001"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2026,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Focal adhesions are dynamic multi\u2010protein complexes that mechanically link the actin cytoskeleton to the extracellular matrix through integrin receptors. A single migrating cell can contain &gt;1000 adhesion structures simultaneously, exhibiting highly heterogeneous fates: only \u223c3.5% of nascent adhesions mature into focal contacts or focal adhesions, while the majority turn over within minutes. Resolving this heterogeneity requires automated detection, tracking, and classification of individual adhesion trajectories at scale. Here we provide a step\u2010by\u2010step Basic Protocol for the Focal Adhesion Package, a MATLAB\u2010based pipeline that processes time\u2010lapse fluorescence images through 11 sequential steps, from sub\u2010pixel stage drift correction and cell masking through point source detection, particle tracking, focal adhesion segmentation, feature extraction, and machine learning\u2010based classification into 9 functional groups. The pipeline optionally integrates traction force microscopy and multi\u2010channel fluorescence data to perform event detection of significant signal rise and compute temporal cross\u2010correlations between protein recruitment and force generation at individual adhesions. For a typical 100\u2010frame movie, total runtime is \u223c40 min using CPU\u2010parallelized optimization (\u223c15 min for preprocessing and detection, \u223c25 min for feature extraction) on a standard workstation, with GPU acceleration reducing this to \u223c27 min. We describe parameter selection criteria for each step and provide supplementary code recipes for programmatic access to outputs. The pipeline can be executed through a graphical user interface or automated using MATLAB scripts. \u00a9 2026 Wiley Periodicals LLC.<\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol<\/jats:bold>\n                    : Software installation and set up\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol<\/jats:bold>\n                    : Running the Focal Adhesion Package\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Alternate Protocol<\/jats:bold>\n                    : Batch processing multiple movies\n                  <\/jats:p>","DOI":"10.1002\/cpz1.70420","type":"journal-article","created":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T16:15:29Z","timestamp":1784045729000},"source":"Crossref","is-referenced-by-count":0,"title":["High\u2010Throughput Focal Adhesion Analysis Software for Automated Detection, Tracking, and Classification of Cell\u2010Matrix Adhesions"],"prefix":"10.1002","volume":"6","author":[{"given":"Majid","family":"Rostami","sequence":"first","affiliation":[{"name":"Department of Biomedical Engineering Michigan Technological University  Houghton Michigan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1384-665X","authenticated-orcid":false,"given":"Sangyoon J.","family":"Han","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering Michigan Technological University  Houghton Michigan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,7,14]]},"reference":[{"key":"e_1_2_9_2_1_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2014.07.011"},{"key":"e_1_2_9_2_2_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0022025"},{"key":"e_1_2_9_2_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncb1763"},{"key":"e_1_2_9_2_4_1","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.66151"},{"key":"e_1_2_9_2_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.3430"},{"key":"e_1_2_9_2_6_1","doi-asserted-by":"publisher","DOI":"10.5483\/BMBRep.2020.53.2.308"},{"key":"e_1_2_9_2_7_1","doi-asserted-by":"publisher","DOI":"10.1021\/acsnano.4c03214"},{"key":"e_1_2_9_2_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.1237"},{"key":"e_1_2_9_2_9_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature09621"},{"key":"e_1_2_9_2_10_1","doi-asserted-by":"publisher","DOI":"10.1002\/cpz1.233"},{"key":"e_1_2_9_2_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrm2957"},{"key":"e_1_2_9_2_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2012.11.034"},{"key":"e_1_2_9_2_13_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2525452123"},{"key":"e_1_2_9_2_14_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncb0807\u2010858"},{"key":"e_1_2_9_3_2_1","unstructured":"https:\/\/github.com\/HanLab\u2010BME\u2010MTU\/focalAdhesionPackage"},{"key":"e_1_2_9_3_4_1","unstructured":"https:\/\/doi.org\/10.5281\/zenodo.19026532"}],"container-title":["Current Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70420","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cpz1.70420","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70420","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T14:33:08Z","timestamp":1785853988000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpz1.70420"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7]]},"references-count":16,"journal-issue":{"issue":"7","published-print":{"date-parts":[[2026,7]]}},"alternative-id":["10.1002\/cpz1.70420"],"URL":"https:\/\/doi.org\/10.1002\/cpz1.70420","archive":["Portico"],"relation":{},"ISSN":["2691-1299","2691-1299"],"issn-type":[{"value":"2691-1299","type":"print"},{"value":"2691-1299","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7]]},"article-number":"e70420"}}