Combined single molecule microscopy

The main goal of the present project is to combine two established high-end methods of single-molecule spectroscopy, namely atomic force microscopy (AFM) and wide-field fluorescence microscopy (WFM), for studying the response of T-cells to the localized and controlled stimulation of specific membrane receptors. AFM allows for the localization of individual molecules with unprecedented spatial resolution in the sub nanometer range, and for the identification of the localized molecules by force spectroscopy (measuring interaction forces of specific binding between target molecule and a probe molecule bound to the AFM probe tip). Moreover, AFM can be used for directed and highly localized binding to specific molecular receptors. Single molecule fluorescence detection with WFM is an excellent and ultimate sensitive technique to monitor individual tagged molecules on and within live cells, thus allowing for the study of rearrangements of cellular structures on the single molecule level, and to monitor interactions between different target molecules. The combination of both microscopy techniques opens completely new horizons for single cell studies. The AFM allows for the localization of specific receptor molecules on a cell surface (receptor mapping), and for a subsequent exactly timed and localized stimulation of a defined number of receptors. After stimulation, the single molecule sensitive fluorescence imaging with WFM allows for the monitoring of signal processes following stimulation up to cellular responses (e.g. cell surface rearrangement, receptor conjugation, structural changes of receptor molecules, biochemical transformations, or opening of ion channels) with highest temporal (on a millisecond time scale) and spatial (on a 100 nm distance scale) resolution. T-cells are a key player in the immune response of living organisms. The study of their response to external stimuli and the elucidation of the necessary conditions for triggering their immunological functions is of great interest from a basic science point of view, and of crucial importance for many applications in medical immunology. Thus, we have chosen the study of T-cell response as the biological test system for applying the proposed AFM/WFM set-up. With the proposed project, we hope to reach a new quality level in the study of the molecular and cell biology of living cells. The combination of AFM and single molecule sensitive WFM is not merely a simple addition of two components. It will provide a qualitatively new level in microscopic studies, giving unprecedented versatility in the detection and monitoring of cellular events with highest spatial and temporal resolution.

The specific activation of T cells represents one key step in the regulation of the immune response. Antigens presented by professional cells are identified by T cells, and initiate a diversity of responses: while agonists lead to T cell activation, antagonists drive the T cell into anergy. For precise regulation of the antigen recognition, a stable contact zone between T cell and antigen-presenting cell is formed, which is called the immunological synapse. In this synapse, receptor and co-receptor molecules organize in a concerted fashion, which appears critical for the proper immune response. The decisive step represents the binding of the T cell receptor to the presented antigen molecule. Not much data are available yet which describe this critical process, mainly due to a lack of proper technologies. In this project, we therefore developed novel technologies which enable the study of T cell activation at the level of single biomolecules. Together with our collaboration partners in Vienna and Stanford, we established protocols for activating T cells on a microscope-based platform, opening up the venue for high-resolution imaging of this process. Single fluorescently labeled biomolecules were imaged, as they move within the complex environment of the immunological synapse. The paths of individual molecules were analyzed to gain insights into heterogeneities in the local environment of the diffusing molecule, which is currently a hot topic in cell biology: we found out that nanometer-sized lipid domains are much more heterogeneous than expected. Single molecule trajectories contain additional information about the binding probability between the T cell receptor and the antigen presented via MHC class II molecules. We succeeded to measure for the first time this interaction process directly in the synapse, which provides now access to physical parameter for theoretical modeling of the activation process. Moreover, the comparative analysis of various different peptides allows for identifying, which physical property is decisive for the antigen recognition process. In addition to performing single molecule experiments on living T cells, we developed novel variants of this technology in order to fully utilize the capabilities of the single molecule approach to explore the nanoscopic organization of living cells. First, we combined optical and force microscopy to enable the defined stimulation of T cells via a single antigen molecule, with the simultaneous optical read-out of the cellular response. Second, a novel method for measuring molecular associations in the cellular plasma membrane was developed. As dimerization and organization to larger structures represent a pivotal step in many signaling pathways, we expect this methodology to become a well-received tool for the scientific community.