Scanning FCS and Superresolution Microscopy on 2D Lipid Membranes

Abstract

Fluorescence Correlation Spectroscopy (FCS) is an established tool for understanding the dynamics of complex cellular processes. By applying the approach scanning FCS (sFCS), significant improvements can be obtained when studying slowly diffusing species, as is often the case in cell membranes. In sFCS, the excitation volume is scanned rapidly through the sample allowing for the simultaneous measurement of the diffusion of species at multiple locations within the sample. This significantly increases the statistical accuracy. In addition, shorter residence times of the fluorophores lead to lower photon doses experienced by each detected molecule, reducing the risk of photobleaching. This is especially important for sensitive fluorophores or when performing combined Stimulated Emission Depletion (STED) and FCS measurements. An added advantage of the scanning process is the ability to determine the observation volume without prior calibration. Here, we show results obtained with a confocal time-resolved fluorescence microscope (MicroTime 200 STED equipped with a FLIMbee galvo scanner, PicoQuant). We performed sFCS measurements on supported lipid bilayers (SLBs) which are commonly used as a simple model system for biological membranes. As the thickness of a bilayer is just a few nanometers, the diffusion properties determined with FCS or sFCS usually correspond to an average over both leaflets. We utilize the fluorescence lifetime information in order to achieve an axial nanometric localization based on Metal Induced Energy Transfer (MIET) [Karedla et al. (2014).ChemPhysChem 15(4):705-711]. The measured lifetime values enable us to separate the different molecular diffusion properties within the upper and the lower leaflet of the SLB measured on graphene.