Advanced Optical Methods in Cell Biology

描述(申请人提供)：项目概要/摘要在我们开发超灵敏、高分辨率、基于液晶的偏光显微镜(LC-PolScope)的基础上，我们现在正在扩大项目的范围，包括激光显微手术和荧光各向异性测量，以开发先进的光学方法来操纵和分析协调和维持细胞分裂的动态细胞结构。1.研制了一种基于固体激光器和液晶空间光调制器的多功能激光显微手术工具。我们将使用基于波前相位调制的先进光束整形光学技术来产生可调的烧蚀图案，以改善细胞内的焦点，并动态塑造和定位焦点区域，以适应细胞结构和功能状态的变化。2.利用激光显微外科手术工具，分析微管阵列在细胞主动分裂过程中的组装/拆解机制。结合LC-PolScope，我们将以高空间和时间分辨率记录星体阵列、动粒体束和极间纺锤体MT中MT丢失和重新生长的结构后果，揭示区分这些微管类别的动态特性。3.我们建议开发一个成像系统，用于快速和全面地分析在表达荧光标记的(例如，GFP标记的)细胞分裂蛋白的细胞中观察到的荧光各向异性。目标是对荧光标记的蛋白质进行全面的光谱和偏振分析，重点是细胞分裂所必需的分子聚集体的动态组织。4.用于测量荧光各向异性的仪器将应用于两个模型：(1)酵母细胞中沙漏和位于芽颈的环结构中隔膜细丝的动态组织；(2)动粒蛋白的结构-功能关系，其自然的张力和释放周期对所有真核细胞都是重要的；对于后者，将使用标记了GFP的酵母NDC80进行初步工作。公共卫生相关性：项目叙述与之前的赠款时期一样，在这一时期开发的显微镜仪器将在医学研究和临床环境中得到应用。此外，这笔赠款下即将进行的细胞生物学应用项目将对我们理解细胞分裂具有广泛的影响，细胞分裂是健康和患病组织中的一个基本细胞过程。

DESCRIPTION (provided by applicant): PROJECT SUMMARY/ABSTRACT Building on our development of the ultra-sensitive and high resolution, liquid crystal based polarizing microscope (LC-PolScope), we are now broadening the scope of the project to include laser microsurgery and fluorescence anisotropy measurements for the development of advanced optical methods to manipulate and analyze the dynamic cell architecture that orchestrates and sustains cell division. 1. We propose to develop a versatile laser microsurgery tool based on a solid-state laser and a liquid crystal spatial light modulator. We will use advanced beam shaping optics based on wave front phase modulation to generate ablation patterns that can be tuned to improve the focus inside a cell and to dynamically shape and position the focal region to adapt to changing cell structure and functional state. 2. Using the laser microsurgery tool we will analyze the assembly/disassembly mechanisms of microtubule (MT) arrays in actively dividing cells. In combination with the LC-PolScope we will record at high spatial and temporal resolution the structural consequences of MT loss and regrowth in the astral arrays, kinetochore bundles and interpolar spindle MTs, shedding light on the dynamic properties that distinguish these microtubule classes. 3. We propose to develop an imaging system for fast and comprehensive analysis of fluorescence anisotropy observed in cells expressing fluorescently labeled (e.g. GFP- tagged) cell division proteins. The goal will be comprehensive spectral and polarization analysis of fluorescently labeled proteins with emphasis on dynamic organization of molecular aggregates essential for cell division. 4. The instrumentation for measuring fluorescence anisotropy will be applied to two models: (1) dynamic organization of septin filaments in the hourglass and ring structures located at the bud neck in yeast cells; and (2) structure-function relationship of kinetochore proteins whose natural cycles of tension and release are important to all eukaryotic cells; for the latter, initial work will be done using yeast GFP tagged Ndc80, a ubiquitous kinetochore protein. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE As in previous grant periods, the microscopy instrumentation developed under this period will have applications in medical research and clinical settings. Furthermore, the cell biological application projects that will be forthcoming under this grant will have broad implications for our understanding of cell division, a fundamental cellular process in healthy and diseased tissue.