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Christian Eggeling


Nano-Immunology

The main research interests of my laboratory are focused on the application and development of ultra-sensitive, live-cell fluorescence microscopy techniques with a spatial resolution down to the molecular level (super-resolution microscopy or nanoscopy), superior to conventional optical microscopes. These super-resolution microscopes will be used to unravel nanoscopic changes at the molecular level in living cells following cellular immune responses. We are planning to visualize previously un-detectable molecular interactions (such as protein-protein and protein-lipid interactions), which will shed new light on different molecular pathways triggered at the cell surface and intracellularly during antigen presentation by dendritic cells and T cell activation. A list of ongoing and future projects is summarized below:

Breaking the resolution limit of conventional microscopy

Studies of complex biological systems such as of living cells demand the use of non-invasive and very sensitive analysis technique such as far-field fluorescence microscopy. Its only drawback is the limited spatial resolution: the diffraction of light prevents that objects closer than about 200 nm can be discerned. As a consequence, important details of for example the cellular immune response cannot be disclosed. A remedy to this physical limit is the on-off switching of fluorescence, ensuring that the measured signal stems from a region of the sample that is much smaller than these 200 nm. Examples of such super-resolving microscopes or nanoscopes are based on Stimulated Emission Depletion (STED) far-field microscopy, on the use of photoswitchable fluorescent markers (RESOLFT or PALM/STORM/… microscopy), or on the optical shelving into the fluorescence marker’s dark state (GSD(IM) microscopy). These techniques deliver a spatial resolution of down to below 40 nm in the living cell and, as a consequence, details of cellular structures and protein aggregations can be imaged and analyzed with much larger details. We apply and develop these techniques further to get new insights into different immunological processes.

Single-molecule super-resolution microscopy of membrane dynamics

Many cellular responses lead to subtle changes on the molecular level, demanding not only for a superior spatial resolution of the analyzing method but also for the sensitivity to monitor single molecules over time and space. The combination of STED microscopy with single-molecule sensitive fluorescence-detection tools such as Fluorescence Correlation Spectroscopy (FCS) as well as the fast spatio-temporal tracking of single labeled molecules (single-particle tracking, SPT) allows for the disclosure of complex dynamical processes otherwise impeded by the limited spatial resolution of conventional far-field microscopy. For example, STED-FCS or SPT offered us to gain novel insights into important cellular processes, such as lipid-lipid, lipid-protein, and protein-protein interactions and the formation of so-called “lipid-rafts” in the cellular plasma membrane. These molecular interactions play an important role in the cellular immune response. We will therefore apply and further develop the STED-FCS and SPT nanoscopy techniques to highlight important molecular processes on the plasma membrane as well as inside the cell during immunological reactions.

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