T cell activation begins with the formation of signalling complexes at the cell surface involving the T cell antigen receptor (TCR), the Src family kinase Lck and the adaptor protein, linker for activation of T cells (LAT).How early TCR signalling events are regulated to prevent inopportune signalling in resting T cells but efficient activation upon receptor ligation is poorly understood.We have established single molecule localization microscopy to determine how TCR engagement reorganizes signalling proteins on the molecular scale.Imaging single molecules in intact cells has provided new insights into the mechanisms of Lck clustering (Rossy et al.Nat Immunol 2013) and LAT recruitment (Williamson et al.Nat Immunol 2011) upon TCR activation.We are now extending this work to elucidate how the membrane environment (Owen et al.Nat Commun 2012) and topography (Owen et al.Biophys J 2013) affects protein interactions to better understand the molecular principles of TCR signalling regulation.