Heterogeneous diffusion dynamicsof molecules play an important role in many cellular signaling events,such as of lipids in plasma membrane bioactivity. However, these dynamicscan often only be visualized by single-molecule and super-resolutionoptical microscopy techniques. Using fluorescence lifetime correlationspectroscopy (FLCS, an extension of fluorescence correlation spectroscopy,FCS) on a super-resolution stimulated emission depletion (STED) microscope,we here extend previous observations of nanoscale lipid dynamics inthe plasma membrane of living mammalian cells. STED-FLCS allows animproved determination of spatiotemporal heterogeneity in moleculardiffusion and interaction dynamics via a novel gated detection scheme,as demonstrated by a comparison between STED-FLCS and previous conventionalSTED-FCS recordings on fluorescent phosphoglycerolipid and sphingolipidanalogues in the plasma membrane of live mammalian cells. The STED-FLCSdata indicate that biophysical and biochemical parameters such asthe affinity for molecular complexes strongly change over space andtime within a few seconds. Drug treatment for cholesterol depletionor actin cytoskeleton depolymerization not only results in the alreadypreviously observed decreased affinity for molecular interactionsbut also in a slight reduction of the spatiotemporal heterogeneity.STED-FLCS specifically demonstrates a significant improvement overprevious gated STED-FCS experiments and with its improved spatialand temporal resolution is a novel tool for investigating how heterogeneitiesof the cellular plasma membrane may regulate biofunctionality. [ABSTRACT FROM AUTHOR]