Biophysikalische Bildgebung

The understanding of the plasma membrane nano-scale organisation in living cells requires microscopy techniques with high temporal and spatial resolution that allow for the quantification of membrane biophysical properties. Among the most popular super-resolution techniques, stimulated emission depletion (STED) microscopy offers high temporal resolution. However, monitoring live processes using STED microscopy is limited by photobleaching. A remedy are exchangeable membrane dyes that only temporarily reside in the membrane. Here, we show that NR4A, a polaritysensitive exchangeable plasma membrane dye, permits the super-resolved quantification of membrane biophysical parameters in real time. STED-fluorescence correlation spectroscopy (STED-FCS) enables the simultaneous quantification of membrane dynamics and lipid packing, which correlate in model and live cell membranes. To showcase potential applications of polarity-sensitive exchangeable dyes, we use live 3D-STED imaging to monitor (i) the formation of giant plasma membrane vesicles, and (ii) lipid exchange during membrane fusion.