Differential Force Microscopy

描述(申请人提供)：真核细胞在与环境相互作用并对内部和外部信号做出反应时，会经历动态的、精心安排的形状变化。例如，中性粒细胞挤过血管壁，吞噬不受欢迎的细菌，只有在子细胞物理分离后，细胞分裂才能完成。如果没有对运动本身的详细了解，就不能完全理解为这些极其复杂的细胞运动提供动力和控制的分子网络。 这一建议开发并展示了一种差力显微镜(DFM)，它克服了原子力显微镜(AFM)的单悬臂限制，能够从根本上对细胞运动进行新的生物物理测量。原子力显微镜最初是用来测量无生命样品的表面性质，而不是跟踪动态细胞的复杂运动。商业上可用的AFM使用单个悬臂一次测量一个点，防止在不同点提供瞬时比较，并禁止对仪器漂移的测量进行实时校正。在所提出的仪器中，通过同时操作两个独立的悬臂，克服了对动态细胞运动的机械测量的这一限制。 这项工作的目的是依次开发三种特定的测量能力，这将广泛地有助于细胞运动的生物物理研究：(1)通过归一化仪器漂移来测量细胞表面的绝对运动，(2)同时测量施加在细胞表面上的两个不同点的力，以及(3)刺激细胞上的一个点并在第二个点测量其机械响应。对于每个目标，都对鱼类角质细胞进行了演示测量，这是细胞运动的一个模型系统。由此产生的仪器将提供给对量化细胞运动的空间和时间协调感兴趣的研究人员。

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.