In recent years, great effort has been made by scientists on developing methods to unveil the basic principles governing life. Among them, single particle tracking (SPT) has become a wide-spread approach as it allows for tracking organelles or even single molecules, which can help to decipher their function in the complex cell machinery. Recent focus has been to push the limits towards faster and smaller scales for detection. In this thesis, we study the diffusion of lipids, which are the major constituents of cell membranes. We present tracking of single gold nanoparticles (GNPs) bound to lipids in model membrane systems using interferometric scattering microscopy (iSCAT). For the first time, imaging speeds up to 1 MHz and a localization precision of a few nanometers are demonstrated, far surpassing existing fluorescence-based methods. Moreover, the detection sensitivity is pushed to the tracking of GNPs with 5nm in diameter, which drastically diminishes the major concern in single particle tracking as to whether the large label perturbs the motion of the molecule under study.
Thus, iSCAT presents a measurement strategy for precise and detailed studies of lipid diffusion.
Model membranes are widely employed for investigating membrane phenomena as they provide a more controlled environment compared to the high complexity found in cell membranes. To fully understand these systems, a precise and detailed characterization is necessary for a correct interpretation of their behavior and properties. In this thesis, measurements are carried out for a range of model membrane systems: supported lipid bilayers (SLBs), pore-spanning membranes (PSMs) and giant unilamellar vesicles (GUVs). We study the diffusion behavior of DOPE lipids in a DOPC lipid membrane in all three different model systems.
The experimental and analytical routine established during the course of this work paves the way towards a plethora of experiments addressing biomedical and biophysical questions.