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The data generated by this method can be overwhelming and its analysis, even for the skilled microscopist, is tedious and error\u2010prone. We developed <jats:styled-content style=\"fixed-case\">SpatTrack<\/jats:styled-content>, an open source, platform\u2010independent program collecting a variety of methods for analysis of vesicle dynamics and distribution in living cells. <jats:styled-content style=\"fixed-case\">SpatTrack<\/jats:styled-content> performs <jats:styled-content style=\"fixed-case\">2D<\/jats:styled-content> particle tracking, trajectory analysis and fitting of diffusion models to the calculated mean square displacement. It allows for spatial analysis of detected vesicle patterns including calculation of the radial distribution function and particle\u2010based colocalization. Importantly, all analysis tools are supported by Monte Carlo simulations of synthetic images. This allows the user to assess the reliability of the analysis and to study alternative scenarios. We demonstrate the functionality of <jats:styled-content style=\"fixed-case\">SpatTrack<\/jats:styled-content> by performing a detailed imaging study of internalized fluorescence\u2010tagged Niemann Pick <jats:styled-content style=\"fixed-case\">C2<\/jats:styled-content> (<jats:styled-content style=\"fixed-case\">NPC2<\/jats:styled-content>) protein in human disease fibroblasts. Using <jats:styled-content style=\"fixed-case\">SpatTrack<\/jats:styled-content>, we show that <jats:styled-content style=\"fixed-case\">NPC2<\/jats:styled-content> rescued the cholesterol\u2010storage phenotype from a subpopulation of late endosomes\/lysosomes (<jats:styled-content style=\"fixed-case\">LE<\/jats:styled-content>\/<jats:styled-content style=\"fixed-case\">LYSs<\/jats:styled-content>). This was paralleled by repositioning and active transport of <jats:styled-content style=\"fixed-case\">NPC2<\/jats:styled-content>\u2010containing vesicles to the cell surface. The potential of <jats:styled-content style=\"fixed-case\">SpatTrack<\/jats:styled-content> for other applications in intracellular transport studies will be discussed.<\/jats:p><jats:p><jats:inline-graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"graphic\/tra12228-gra-0001.png\" xlink:title=\"image\"\/><\/jats:p>","DOI":"10.1111\/tra.12228","type":"journal-article","created":{"date-parts":[[2014,9,22]],"date-time":"2014-09-22T10:35:39Z","timestamp":1411382139000},"page":"1406-1429","source":"Crossref","is-referenced-by-count":35,"title":["<scp>SpatTrack<\/scp>: An Imaging Toolbox for Analysis of Vesicle Motility and Distribution in Living Cells"],"prefix":"10.1111","volume":"15","author":[{"given":"Frederik W.","family":"Lund","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Biology University of Southern Denmark  DK\u20105230 Odense M Denmark"},{"name":"Department of Biochemistry Weill Medical College of Cornell University  York Ave. 1300 10065 NY USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria Louise V.","family":"Jensen","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology University of Southern Denmark  DK\u20105230 Odense M Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tanja","family":"Christensen","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology University of Southern Denmark  DK\u20105230 Odense M Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gitte K.","family":"Nielsen","sequence":"additional","affiliation":[{"name":"Department of Biomedicine University of Aarhus  DK\u20108000 Aarhus C. 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