3D nanoscopy of the immunological synapse

This project aims at obtaining a deeper understanding of the functioning of the adaptive immune system, in particular its outstanding capability to discriminate friend from foe: sometimes a single harmful molecule is sufficient to trigger an immune response. This exquisite sensitivity is not fully understood to date. Crucial for a better understanding will be the direct and careful observation of the communication between cells of the adaptive immune system (T-cells) and those cells which collect potentially harmful molecules and present them to the T-cells upon direct contact. To investigate the complex molecular interaction at these contact sites spatially and temporarily, we will improve and finally combine three of the most precise measurement techniques available to date in a single microscope. We anticipate that our approach will enable measuring 3D molecule positions at accuracies down to 10 nm, which corresponds to a length of merely about 100 atom diameters. This sensitivity will enable to follow the communication between cells at unprecedented accuracy and might thus reveal important unknown details of the inter-cellular communication.

Our interdisciplinary project aims to find out more about antigen recognition by T cells, special lymphocytes of our immune system. T cells interact with other cells in our body (antigen-presenting cells), which present tiny fragments of foreign substances (e.g. from viruses) to the T cells. It is currently not clear exactly how potentially dangerous foreign substances can be recognized so effectively by T cells and subsequently trigger an immune response of the entire body. We have been able to achieve results in two fields of research: Microscopy Here, technological aspects of nanoscopy, i.e. microscopy on the smallest size scales, were further developed. A simple method was developed to determine 3D positions of individual bio-molecules - for example proteins in the cell membrane - with an accuracy of only a few nanometers. In our research project, this method was used to find out more about antigen recognition by T cells. Furthermore, our research could answer the question whether a commonly established method (interference reflection microscopy, IRM) can be used to reliably determine the distances of a cell membrane from the microscope coverslip. We were able to show that IRM works reliably most of the time, but provides serious errors in some areas of the cell membrane. Biology With respect to biological aspects, our research was able to answer the following main questions: 1) Do "nanoclusters" of T cell receptors (TCR) really exist? We were able to demonstrate that the previously described TCR nano-clusters in the cell membrane of inactive T cells can be attributed to artifacts of imaging and data analysis. Some dye molecules are counted multiple times, thus fooling locally higher TCR densities - i.e. TCR clusters. 2) What are physical reasons for the observation of TCR - microclusters? The observation of microclusters during T cell activation could also be partly due to imaging artifacts: Being closer to the coverslip, these molecules appear particularly bright due to increased light coupling into the coverslip, which in turn may suggest a molecule enrichment. We were able to show that this "enhancement effect" plays only a minor role and that molecular enrichment, i.e. the formation of micro-clusters, actually takes place. 3) Is the distance between the membranes of T cell and antigen-presenting cell too small for the protein CD45? An established theory for triggering T cells involves the large molecule CD45. Therefore, it is important to know whether this molecule fits into the narrow gap between the two cell membranes at all. Our highly accurate measurements suggest that the membrane gap might indeed be too small for CD45, both for active and inactive T cells. This now allows us to narrow down the possible theories for T cell activation.