title: High-Content Microscopy Analysis of Subcellular Structures: Assay Development and Application to Focal Adhesion Quantification.
abstract: High-content analysis (HCA) converts raw light microscopy images to quantitative data through the automated extraction, multiparametric analysis, and classification of the relevant information content. Combined with automated high-throughput image acquisition, HCA applied to the screening of chemicals or RNAi-reagents is termed high-content screening (HCS). Its power in quantifying cell phenotypes makes HCA applicable also to routine microscopy. However, developing effective HCA and bioinformatic analysis pipelines for acquisition of biologically meaningful data in HCS is challenging. Here, the step-by-step development of an HCA assay protocol and an HCS bioinformatics analysis pipeline are described. The protocol's power is demonstrated by application to focal adhesion (FA) detection, quantitative analysis of multiple FA features, and functional annotation of signaling pathways regulating FA size, using primary data of a published RNAi screen. The assay and the underlying strategy are aimed at researchers performing microscopy-based quantitative analysis of subcellular features, on a small scale or in large HCS experiments. © 2016 by John Wiley & Sons, Inc.
authors.affiliation: Leibniz Institute on Aging-Fritz Lipmann Institute, Jena, Germany.|These authors contributed equally to this work.
authors.forename: Torsten
authors.initials: T
authors.lastname: Kroll
authors.affiliation: Leibniz Institute on Aging-Fritz Lipmann Institute, Jena, Germany.|Current address: Max Planck Institute for Molecular Biomedicine, Münster, Germany.|These authors contributed equally to this work.
authors.forename: David
authors.initials: D
authors.lastname: Schmidt
authors.affiliation: Leibniz Institute on Aging-Fritz Lipmann Institute, Jena, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Schwanitz
authors.affiliation: Leibniz Institute on Aging-Fritz Lipmann Institute, Jena, Germany.|Institute for Comparative Molecular Endocrinology, University of Ulm, Ulm, Germany.
authors.forename: Mubashir
authors.initials: M
authors.lastname: Ahmad
authors.affiliation: Leibniz Institute on Aging-Fritz Lipmann Institute, Jena, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Hamann
authors.affiliation: Leibniz Institute on Aging-Fritz Lipmann Institute, Jena, Germany.
authors.forename: Corinne
authors.initials: C
authors.lastname: Schlosser
authors.affiliation: Leibniz Institute on Aging-Fritz Lipmann Institute, Jena, Germany.
authors.forename: Yu-Chieh
authors.initials: YC
authors.lastname: Lin
authors.affiliation: Leibniz Institute on Aging-Fritz Lipmann Institute, Jena, Germany.
authors.forename: Konrad J
authors.initials: KJ
authors.lastname: Böhm
authors.affiliation: Institute for Comparative Molecular Endocrinology, University of Ulm, Ulm, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Tuckermann
authors.affiliation: Leibniz Institute on Aging-Fritz Lipmann Institute, Jena, Germany.
authors.forename: Aspasia
authors.initials: A
authors.lastname: Ploubidou
mesh_terms: D000818:Animals; D001331:Automation; D019556:COS Cells; D002452:Cell Count; D002522:Chlorocebus aethiops; D022001:Focal Adhesions / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D034622:RNA Interference; D012984:Software; D013194:Staining and Labeling; D013347:Subcellular Fractions / Q000378:metabolism
keywords: focal adhesions; high-content analysis; high-content screening; microscopy automation; quantitative microscopy; subcellular organelles