Abstract: CD4 positive Helper T cells form an essential component of the adaptive immune response. As such, they are required to activate when required - to ward off infection, and remain inactive when confronted with the body’s own tissues – to avoid autoimmune disease. It is now known that a mechanism partly responsible for this tight regulation is nanoscale protein spatio-temporal organization, especially the nanoclustering of surface proteins. Here, I will outline my lab’s latest work on quantifying the nanoscale organization of protein using super-resolution microscopy via single molecule localization microscopy (SMLM). In particular, I will focus on many-colour quantitative statistical cluster analysis and stochastically accurate DNA-PAINT imaging. These are applied to a negative regulating pathway involving Csk, TRAF3 and PAG which is relevant to autoimmune diseases including rheumatoid arthritis, lupus and diabetes. I will also outline out latest work analysing the spatial organization of membrane lipids using environmentally-sensitive lipid probes and advanced microscopy.