Abstract
T-cells engage with antigen-presenting cells in search for antigenic peptides and form transient interfaces termed immunological synapses. Synapse topography affects receptor binding rates and the mutual segregation of proteins due to size exclusion effects. It is hence important to determine the 3D topography of the immunological synapse at high precision. Current methods provide only rather coarse images of the protein distribution within the synapse. Here, we applied supercritical angle fluorescence microscopy combined with defocused imaging, which allows three-dimensional single molecule localization microscopy (3D-SMLM) at an isotropic localization precision below 15 nm. Experiments were performed on hybrid synapses between primary T-cells and functionalized glass-supported lipid bilayers. We used 3D-SMLM to quantify the cleft size within the synapse by mapping the position of the T-cell receptor (TCR) with respect to the supported lipid bilayer, yielding average distances of 18 nm up to 31 nm for activating and nonactivating bilayers, respectively.
Highlights
Understanding the topography of cellular interfaces is central for addressing many cell biological questions
A common way to study antigen-specific T-cell activation involves the use of functionalized glass-supported lipid bilayers (SLBs) as surrogates of antigen-presenting cell (APC).[5−7] This experimental design has several advantages when it comes to the application of highresolution microscopy techniques while still preserving the essential hallmarks of T-cell signaling including the formation of an immunological synapse, the recruitment of the kinase
In order to evaluate the correlation between 3D-single molecule localization microscopy (SMLM) and interference reflection microscopy (IRM) data, we sought a system with known separation of the detected single dye molecules from the glass surface
Summary
Understanding the topography of cellular interfaces is central for addressing many cell biological questions. The IRM images show substantial contrast fluctuations (Figure 2b), indicating corresponding fluctuations in the distance of the T-cell membrane from the SLB This is supported by the 3D-SMLM data, where the determined TCR z-positions spread between 0 and 300 nm. It is instructive to compare the obtained 3D SMLM and IRM images with conventional diffraction-limited TIR fluorescence microscopy images (Figure 3; see SI supporting gallery Figures 1 and 2 for additional examples) In this particular example, the cell had been fixed just before the formation of the central supramolecular activation cluster (cSMAC), when TCR microclusters were observed in a ringlike structure around the cell center (Figure 3i). The residual separation reflected in all likelihood the distance separating the dye site- conjugated to the single chain antibody fragment and the dye coupled to the MCC peptide’s Cterminus as presented by I-Ek.[6]
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