Combined Atomic Force and Confocal Laser Scanning Microscopy to Investigate Structure-Function-Relationships of Cells and Tissue on the Micrometer and Nanometer Lengthscale

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With the combined atomic force microscope (AFM) and confocal laser scanning microscope (CLSM) setup, we plan to investigate the relationship between structure and biomechanics of the cytoskeleton and the extracellular matrix (ECM), using AFM imaging and indentation measurements (IT-AFM) combined with confocal optical microscopy. We will focus on the effect of biomechanical properties on cell behavior, as well as on structure formation, morphogenesis and pathogenesis of biological tissue. For the planned experiments, the density of IT-AFM data points (force curves) should be sufficiently high, to allow for molecular resolution and for correlating changes in cell and ECM biomechanics directly to the components of the cytoskeleton or the ECM, even for large regions of interest, which cover whole cells or relevant tissue areas (up 100 µm x 100 µm). This requires a state of the art AFM, which allows for the fast recording of large “force volumes” with high spatial resolution, as well as a modern CLSM which allows for the investigation of mammalian tissue and records fluorescence signals also form deeper layers (up to 1 mm) of tissue sections or tissue constructs.In addition to addressing fundamental questions in mechanobiology, we will focus on structural and biomechanical alterations in articular cartilage in degenerative diseases, such as osteoarthritis (OA) and cartilage regeneration via tissue engineering. In addition, we will investigate the role of ECM structure and biomechanics in cancer spreading and metastasis formation. This will not only provide a better understanding of structure-function relationships in healthy and pathological tissue, but also point out new diagnostic and therapeutic strategies. Therefore, in addition to cells and native tissue, we will also investigate engineered tissue constructs. To adapt the material properties of these tissue constructs to native tissue and biological requirements, we will do this immediately after the biofabrication process, as well as after tissue maturation in specially designed bioreactors. Both for tissue degeneration and regeneration and for cancer cell spreading and metastasis formation, also cell-cell and cell-matrix interaction and the transduction of mechanical signals (mechanotransduction) play an important role. We thus plan to also investigate cell-cell and cell-matrix interactions and cell adhesion using an additional AFM head, which is especially designed for single cell force spectroscopy measurements.