Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging

Surface molecules, distributed in diverse patterns and clusters on cell membranes, influence vital functions of living cells. It is therefore important to understand their molecular surface organisation under different physiological and pathological conditions. Here, we present a model-free, quantitative method to determine the distribution of cell surface molecules based on TIRF illumination and super-resolution optical fluctuation imaging (SOFI). This SOFI-based approach is robust towards single emitter multiple-blinking events, high labelling densities and high blinking rates. In SOFI, the molecular density is not based on counting events, but results as an intrinsic property due to the correlation of the intensity fluctuations. The effectiveness and robustness of the method was validated using simulated data, as well as experimental data investigating the impact of palmitoylation on CD4 protein nanoscale distribution in the plasma membrane of resting T cells.