Systems Imaging of the Immune Synapse

Three‐dimensional live cell imaging of the interaction of T cells with antigen presenting cells (APC) visualises the subcellular distributions of signalling 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 signalling. 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 signalling as it occurs inside live cells with resolution in time and space across thousands of cells.