Leaflet-resolved localization and diffusion of molecules within membranes using metal-induced energy transfer

Diffusion is the most fundamental process of molecular transport in cell membranes and an important factor in maintaining its fluidity, controlling the dynamics and functioning of the membrane. In-depth knowledge of membrane dynamics and its structure is essential for understanding the functional role of membranes and all processes involving them. Therefore, a precise quantitative characterisation of diffusion in membranes is important. While Many different techniques have given insight into this matter, but they fail in addressing the dual-leaflet nature of a membrane. The experimental challenge is the extremely small distance of $\sim$5 nm between the leaflets which is by two orders of magnitude smaller than the diffraction-limited optical resolution of a microscope (ca. 500 nm along the optical axis, for a well-adjusted confocal microscope). In this thesis, I present a new method of super-resolution microscopy that allows me to distinguish between individual leaflets of a Supported lipid bilayer (SLB) and to measure axial distances with nanometer accuracy. Moreover, it allows me to measure the diffusion of lipids in an SLB with a leaflet-resolved manner. Such SLBs serve as simple model systems to study membrane dynamics, and our method will allow me to see the interaction between the leaflets and the effect of an SLBs proximity to the substrate. For this purpose, I combine Metal-induced energy transfer (MIET) with scanning Fluorescence Lifetime Correlation Spectroscopy (sFLCS). MIET exploits the strongly distance-dependent quenching of fluorophores by a nearby metal(or graphene) layer. On replacing the metal layer with a single sheet of graphene, the axial resolution of this method can be pushed down to sub-nanometers, due to the much more localized quenching of fluorescence by graphene. This thesis will mainly focus on MIET/GIET and its application to studying membranes. In combination with FLCS, I am able to determine leaflet-specific diffusion coefficients. A major part of this thesis involves leaflet-resolved diffusion studies on model SLBs.2022-05-1