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

Abstract

The topography of the immune synapse plays a key role in the activation of T cells. Diffusion of membrane proteins is often hindered due to steric effects in close cell-cell contacts. A prime example is the exclusion of the phosphatase CD45 from the close-contact zone due to its large extracellular domain. To study exclusion effects, tracking of proteins in all three dimensions is required. Standard optical microscopy provides high localisation precision in lateral dimensions but is limited in the axial direction. 3D single molecule localisation microscopy (3D SMLM) based on defocused imaging achieves a resolution of 10 nm along the axial direction [1]. The method is validated by tracking the diffusion of two nanoprobes (DNA-NP) on lipid embranes. The nanostructures are custom-designed with DNA nanotechnology and are fluorescently labelled at two z-positions differing by 11 nm. With 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, which is in line with the rational design. 3D tracking of nanostructures provides a powerful tool to map out exclusion zones in the immunological synapse with high resolution.