Imaging Actin Dynamics at the Ventral Surface of Live Cells

描述（由申请人提供）：负责寻路的机制在很大程度上尚未被探索。在二维培养中，寻路被定义为形成局部接触所需的腹侧突起。腹侧突起、侧侧突起和粘连形成之间的关系迄今尚未得到充分的研究，部分原因是目前的光学显微镜无法成像活细胞在刺激突起过程中腹侧表面的肌动蛋白聚合动力学。在本应用中，我们描述了光学成像的最新进展如何与现有的生物传感器相结合，以阐明与腹侧突起有关的空间和时间事件的编排顺序及其与焦点接触形成的关系。我们将：1；将观察腹侧细胞表面动力学的成像方法扩展到活细胞。a)开发硬件和软件，使全内反射荧光（TIRF）成像利用脉冲激光的激发。b)开发硬件和软件，使使用TIRF照明的荧光寿命成像显微镜（FLIM）成为可能。c)开发硬件和软件以优化干涉显微镜（IRM）。2. 可视化活细胞的腹突，并确定腹表面的肌动蛋白区室，这些区室有助于腹突活动和局部接触的形成。

DESCRIPTION (provided by applicant): The mechanisms responsible for pathfinding are largely unexplored. Pathfinding is defined in two-dimensional culture as the formation of ventral protrusions that are required for focal contact formation. The reason that the relationships between ventral protrusions, lateral protrusions and adhesion formation have not been adequately studied to date is in part due to the inability of current light microscopes to image actin polymerization dynamics at the ventral surface in live cells during stimulated protrusion. In this application we describe how recent advances in optical imaging may be combined with existing biosensors in order to elucidate the choreographed sequence of spatial and temporal events that are involved in ventral protrusions and their relationship to focal contact formation. We will: 1. Extend imaging methods for observation of ventral cell surface dynamics to live cells. a) Develop hardware and software to enable total internal reflection fluorescence (TIRF) imaging using excitation from a pulsed laser. b) Develop hardware and software to enable fluorescence lifetime imaging microscopy (FLIM) using TIRF illumination. c) Develop hardware and software in order to optimize interference microscopy (IRM). 2. Visualize ventral protrusions in live cells and determine the actin compartments on the ventral surface that contribute to ventral protrusion activity and the formation of local contacts.