Nanoscale studies of membrane dynamics via STED- Fluorescence Correlation Spectroscopy

Abstract

Molecular membrane dynamics in living cells are often obscured from the observer because of the limited spatial resolution of conventional far-field optical microscopy. The superior spatial resolution of stimulated emission depletion (STED) nanoscopy provides new insights into the dynamic processes within the plasma membrane. In combination with the high temporal resolution of fluorescence correlation spectroscopy (FCS), we record and characterize the diffusion of membrane constituents within nanoscale observation areas. In the first part of the thesis, we compare STED-FCS data of various fluorescent lipid analogs and proteins in the plasma membrane of living cells. Our results reveal distinct modes of diffusion which can be differentiated according to the chemical structure of the molecules. Phosphoglycerolipids diffuse freely and only weakly interact with other membrane constituents. Sphingolipids exhibit a strong molecular confinement due to the formation of hydrogen bounds within the ceramide backbone or between large polar head groups. Transmembrane proteins corral in compartments built by the cellular cytoskeleton. We implement Monte-Carlo simulations to support and explain our experimental results. In the second part of the thesis, we enhance the experimental STED-FCS setup by the integration of a fast scanning unit. This newly developed concept not only enables us to perform calibration-free measurements of slowly diffusing particles but also visualizes spatial diffusion heterogeneities along the scan trajectory.