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

本研究利用原子力显微镜（AFM）和共聚焦激光扫描显微镜（CLSM）相结合的方法，结合AFM成像和压痕测量技术（IT-AFM）和共聚焦光学显微镜，研究细胞骨架和细胞外基质（ECM）的结构与力学之间的关系。我们将着重于生物力学性质对细胞行为的影响，以及对生物组织的结构形成，形态发生和发病机制。对于计划的实验，IT-AFM数据点（力曲线）的密度应该足够高，以允许分子分辨率并将细胞和ECM生物力学的变化直接与细胞骨架或ECM的组分相关联，即使是覆盖整个细胞或相关组织区域（高达100 µm x 100 µm）的大区域。这需要最先进的AFM，它允许快速记录具有高空间分辨率的大“力体积”，以及现代CLSM，它允许研究哺乳动物组织并记录荧光信号，也形成更深层。除了解决机械生物学中的基本问题外，我们将着重于退行性疾病中关节软骨的结构和生物力学改变，如骨关节炎（OA）和通过组织工程的软骨再生。此外，我们将研究ECM结构和生物力学在癌症扩散和转移形成中的作用。这不仅可以更好地理解健康和病理组织的结构-功能关系，还可以指出新的诊断和治疗策略。因此，除了细胞和天然组织，我们还将研究工程组织构建体。为了使这些组织构建体的材料特性适应天然组织和生物学要求，我们将在生物制造过程后以及在专门设计的生物反应器中组织成熟后立即进行这项工作。对于组织变性和再生以及癌细胞扩散和转移形成，细胞-细胞和细胞-基质相互作用以及机械信号的转导（机械转导）也起重要作用。因此，我们计划也调查细胞和细胞基质的相互作用和细胞粘附使用额外的AFM头，这是专门设计用于单细胞力谱测量。

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.