Nanoscale organization of the membranes of living cells plays crucial roles in numerous vital processes, including during the activation of T-cells and their formation of the immunological synapse. However, the detailed nature and function of reorganization of lipids during this key initiating event of immune response remain largely unclear. To gain further insight into the mechanisms, we employed two techniques that probe complementary properties of the membranes at the molecular level: STED-FCS to reveal detailed picture of the diffusion of the lipids, and spectral imaging with environment-sensitive membrane probes, i.e. microspectroscopy, to map differences in local molecular order within the lipid bilayer. We correlated both properties with the locations of the key membrane proteins responsible for triggering the activation of T-cells, such as T-cell receptor. In search for the most sensitive description of the membrane order, we systematically compared the established methods of analysis and representation: generalized polarization, lineshape fitting, and spectral phasors.