Long-term superresolution imaging and quantification of live-cell plasma membrane biophysical properties with smart exchangeable dyes

Abstract

The study of biological membranes by fluorescence microscopy is hindered by the resolution limit of conventional microscopy. Superresolution microscopy overcomes this problem, but greatly limits temporal resolution. In the case of STED superresolution microscopy, high photobleaching rates prevent long-term imaging. Here, we employ a new exchangeable dye that reports on the molecular order and dynamics of lipid membranes with no photobleaching-induced signal loss. We demonstrate that this dye is sensitive to lipid packing changes in model and cell membranes and perform simultaneous STED-FCS dynamics and packing measurements.