3D Localisation Microscopy and Single Molecule Tracking Of Membrane-Bound DNA Nanostructures Using Defocused Imaging

Abstract

At the interface of cells, the 3D topography of the bilayer influences biomolecular interactions in several ways, such as by sterically hindering the diffusion of membrane proteins. A prominent example is the close contact zone during T cell activation where the phosphatase CD45 with its large extracellular domain is excluded. To investigate these exclusion effects, it is essential to track individual membrane proteins in 3D. While standard single molecule localisation microscopy (SMLM) offers high localisation precision in lateral dimensions, its performance in the axial z-direction is limited. Here we describe a new version of SMLM based on defocused imaging which allows tracking in 3D and achieves a resolution of 10 nm along the axial direction. The method is validated by tracking two DNA nanoprobes (DNA-NPs) on supported lipid bilayers. DNA-NPs are custom designed using DNA nanotechnology and are labeled with two fluorophores separated by a distance of 11 nm in the z-axis. Using 3D SMLM, we determine a lateral diffusion coefficient of D ≈ 0.3 μm2/s and a relative height difference of Δz = 11.57 ± 1.01 nm. Our findings lay the foundation for high-resolution 3D tracking of molecules, such as CD45, to map out exclusion zones in the immunological synapse.