Diffusion Measurements at the Nanoscale with STED-FCS

Abstract

The cellular plasma membrane, a seemingly chaotic structure composed of thousands of lipids and proteins, empowers cellular life and represents a versatile hub for signalling. Transient interactions of membrane components integrate to large-scale membrane properties such as packing, tension, curvature, or viscosity and reorganise the membrane in response to physiological needs. How do nanoscale interactions and heterogeneities determine cellular plasma membrane properties? This question is not trivial to address with technologies limited by the diffraction of light. Here, we discuss Stimulated Emission Depletion (STED) nanoscopy and its combination with fluorescence correlation spectroscopy (FCS, STED-FCS) as a technique to shed light onto the relevant molecular scales. While imaging with STED allows for tremendous insights into the spatial domain of organisation, the combination of STED with FCS provides increased temporal resolution and yields insights into the nature of the molecular motion on the relevant scales, allowing us to study nanoscale diffusion modes. We provide a comprehensive overview of the field, introduction to the technique and discuss common pitfalls with its implementation as well as alternative methodologies to measure the diffusion modes on the nanoscale.