title: Assessing Mitochondrial Movement Within Neurons: Manual Versus Automated Tracking Methods.
abstract: Owing to the small size of mitochondria and the complexity of their motility patterns, mitochondrial tracking is technically challenging. Mitochondria are often tracked manually; however, this is time-consuming and prone to measurement error. Here, we examined the suitability of four commercial and open-source software alternatives for automated mitochondrial tracking in neurons compared with manual measurements. We show that all the automated tracking tools dramatically underestimated track length, mitochondrial displacement and movement duration, with reductions ranging from 45 to 77% of the values obtained manually. In contrast, mitochondrial velocity was generally overestimated. Only the number of motile mitochondria and their directionality were similar between strategies. Despite these discrepancies, we show that automated tools successfully detected transport alterations after applying an oxidant agent. Thus, automated methods appear to be suitable for assessing relative transport differences between experimental groups, but not for absolute quantification of mitochondrial dynamics. Although useful for objective and time-efficient measurements of mitochondrial movements, results provided by automated methods should be interpreted with caution.
authors.affiliation: Institute for Medical Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany.|Experimental and Clinical Research Center, A joint cooperation between the Charité-Universitätsmedizin Berlin and the Max-Delbrück Center for Molecular Medicine, Berlin, Germany.
authors.forename: Helena
authors.initials: H
authors.lastname: Bros
authors.affiliation: Deutsches Rheuma-Forschungszentrum, a Leibniz Institute, Berlin, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Hauser
authors.affiliation: Experimental and Clinical Research Center, A joint cooperation between the Charité-Universitätsmedizin Berlin and the Max-Delbrück Center for Molecular Medicine, Berlin, Germany.|NeuroCure Clinical Research Center and Clinical and Experimental Multiple Sclerosis Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Friedemann
authors.initials: F
authors.lastname: Paul
authors.affiliation: Deutsches Rheuma-Forschungszentrum, a Leibniz Institute, Berlin, Germany.
authors.forename: Raluca
authors.initials: R
authors.lastname: Niesner
authors.affiliation: Institute for Medical Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany.|Experimental and Clinical Research Center, A joint cooperation between the Charité-Universitätsmedizin Berlin and the Max-Delbrück Center for Molecular Medicine, Berlin, Germany.
authors.forename: Carmen
authors.initials: C
authors.lastname: Infante-Duarte
mesh_terms: D000818:Animals; D057205:Automation, Laboratory / Q000379:methods; D001370:Axonal Transport; D002478:Cells, Cultured; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D008928:Mitochondria / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D059008:Time-Lapse Imaging / Q000379:methods*
keywords: Difference Tracker; Imaris; Volocity; automated tracking; manual tracking; mitochondrial tracking; mitochondrial transport; wrMTrck