Molecular interactions are key in cellular signalling. They are usually ruled by the organization and mobility of the involved molecules. For example, the direct and non-invasive observation of the interactions in the living cell membrane is often impeded by principle limitations of conventional far-fi eld optical microscopes, for example with respect to limited spatio-temporal resolution and information content. Here, we present an advanced optical microscopy study involving tools such super-resolution STED microscopy in combination with spectral imaging and fl uorescence correlation spectroscopy (FCS) or single-molecule tracking on a MINFLUX microscope. We highlight how these approaches can reveal novel aspects of membrane bioactivity such as of the existence and function of potential lipid rafts and during pathogen invasion, but also reveal limitations.