title: Systems Imaging of the Immune Synapse.
abstract: Three-dimensional live cell imaging of the interaction of T cells with antigen-presenting cells (APCs) visualizes the subcellular distributions of signaling intermediates during T cell activation at thousands of resolved positions within a cell. These information-rich maps of local protein concentrations are a valuable resource in understanding T cell signaling. Here, we describe a protocol for the efficient acquisition of such imaging data and their computational processing to create four-dimensional maps of local concentrations. This protocol allows quantitative analysis of T cell signaling as it occurs inside live cells with resolution in time and space across thousands of cells.
authors.affiliation: School of Cellular and Molecular Medicine, University of Bristol, Bristol, BS8 1TD, UK.
authors.forename: Rachel
authors.initials: R
authors.lastname: Ambler
authors.affiliation: Computational Biology Department, School of Computer Science, Carnegie Mellon University, 7723 Gates-Hillman Center, Pittsburgh, PA, 15213, USA.
authors.forename: Xiangtao
authors.initials: X
authors.lastname: Ruan
authors.affiliation: Computational Biology Department, School of Computer Science, Carnegie Mellon University, 7723 Gates-Hillman Center, Pittsburgh, PA, 15213, USA. murphy@cmu.edu.|Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, 15213, USA. murphy@cmu.edu.|Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, 15213, USA. murphy@cmu.edu.|Department of Machine Learning, Carnegie Mellon University, Pittsburgh, PA, 15213, USA. murphy@cmu.edu.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Murphy
authors.affiliation: School of Cellular and Molecular Medicine, University of Bristol, Bristol, BS8 1TD, UK. christoph.wuelfing@bristol.ac.uk.
authors.forename: Christoph
authors.initials: C
authors.lastname: Wülfing
mesh_terms: D000818:Animals; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D054992:Immunological Synapses / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence / Q000379:methods; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology*
keywords: Computational image analysis; Immunological synapse; Live cell imaging; Spatiotemporal distributions; T cell activation