Differential Force Microscopy

描述（由申请人提供）：真核细胞在与其环境相互作用并对内部和外部信号作出反应时经历动态的、精心编排的形状变化。例如，嗜中性粒细胞挤过血管壁并吞噬不受欢迎的细菌，而细胞分裂只有在子细胞物理分离后才完成。如果没有对运动本身的详细了解，就无法完全理解为这些非常复杂的细胞运动提供动力和控制的分子网络。 该提案开发并演示了一种微分力显微镜（DFM），该显微镜克服了原子力显微镜（AFM）的单悬臂局限性，使细胞运动的全新生物物理测量成为可能。AFM最初是为了测量无生命样品的表面特性而开发的，而不是为了跟踪动态细胞的复杂运动。市售的AFM使用单个悬臂来一次测量一个点，从而防止在不同点处提供瞬时比较并且禁止针对仪器漂移的测量的实时校正。在所提出的仪器中，通过同时操作两个独立的杠杆，克服了动态细胞运动的机械测量的这种限制。 这项工作的目的是依次开发三种特定的测量能力，这将广泛有益于细胞运动的生物物理研究：（1）通过标准化仪器漂移测量细胞表面的绝对运动，（2）同时测量施加在细胞表面两个不同点上的力，以及（3）刺激细胞上的一个点并测量其在第二点的机械响应。对于每一个目标，演示测量进行鱼类角膜细胞，细胞运动的模型系统。由此产生的仪器将提供给有兴趣量化细胞运动的空间和时间协调的研究人员。

DESCRIPTION (provided by applicant): Eukaryotic cells undergo dynamic, carefully-orchestrated shape changes as they interact with their environment and respond to internal and external signals. For example, neutrophils squeeze through the walls of blood vessels and engulf unwelcome bacteria, and cell division is only complete after the physical separation of daughter cells. The networks of molecules that power and control these remarkably complex cell movements cannot be fully understood without detailed knowledge of the movements themselves. This proposal develops and demonstrates a Differential Force Microscope (DFM) that overcomes the single-cantilever limitations of Atomic Force Microscopy (AFM) to enable fundamentally new biophysical measurements of cell movements. AFMs were first developed to measure surface properties of inanimate samples - not to follow the complex movements of dynamic cells. Commercially-available AFMs use a single cantilever to measure one point at a time, preventing instantaneous comparison offered at different points and prohibiting real-time correction of measurements for instrument drift. This limitation on mechanical measurements of dynamic cell movements is overcome in the proposed instrument by operating two independent cantilevers simultaneously. The Aims of this work sequentially develop three specific measurement capabilities that will broadly benefit biophysical studies of cell movements: (1) measure absolute movements of a cell surface by normalizing instrument drift, (2) simultaneously measure forces exerted at two different points on a cell surface, and (3) stimulate one point on a cell and measure its mechanical response at a second point. For each aim, demonstration measurements are performed on fish keratocyte cells, a model system of cell motility. The resulting instrument will be made available to researchers interested in quantifying spatial and temporal coordination of cell movements.