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

结合原子力显微镜(AFM)和激光共聚焦扫描显微镜(CLSM)，我们计划利用原子力显微镜成像和压痕测量(IT-AFM)结合共聚焦光学显微镜来研究细胞骨架和细胞外基质(ECM)的结构和生物力学之间的关系。我们将重点介绍生物力学特性对细胞行为的影响，以及生物组织的结构形成、形态发生和发病机制。对于计划中的实验，IT-AFM数据点(力曲线)的密度应该足够高，以实现分子分辨率，并将细胞和细胞外基质生物力学的变化直接与细胞骨架或细胞外基质的组件关联起来，即使是覆盖整个细胞或相关组织区域的大片感兴趣区域(高达100微米x 100微米)。这需要先进的原子力显微镜(AFM)和现代激光共聚焦显微镜(CLSM)，原子力显微镜可以快速记录高空间分辨率的大“力体积”，现代激光共聚焦显微镜可以研究哺乳动物组织并记录荧光信号，还可以形成更深层次(高达1毫米)的组织切片或组织结构。除了解决机械生物学中的基本问题外，我们还将专注于退行性疾病中关节软骨的结构和生物力学变化，如骨关节炎(OA)和通过组织工程进行的软骨再生。此外，我们还将探讨ECM结构和生物力学在肿瘤扩散和转移形成中的作用。这不仅有助于更好地了解健康和病理组织中的结构-功能关系，而且还将指出新的诊断和治疗策略。因此，除了细胞和天然组织外，我们还将研究工程化组织结构。为了使这些组织结构的材料特性适应本地组织和生物要求，我们将在生物制造过程之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。无论是对于组织的退化和再生，还是对于癌细胞的扩散和转移的形成，细胞-细胞和细胞-基质的相互作用以及机械信号的转导(机械转导)都起着重要的作用。因此，我们计划使用额外的AFM头来研究细胞-细胞和细胞-基质的相互作用和细胞黏附，这是专门为单细胞力谱测量而设计的。

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.