Advanced Optical Methods in Cell Biology

描述（由申请人提供）：项目概要/摘要建立在我们开发的超灵敏和高分辨率，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，以包括激光显微手术和荧光各向异性测量，用于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.我们提出了一个多功能的激光显微手术工具的基础上的固体激光器和液晶空间光调制器。我们将使用基于波前相位调制的先进光束成形光学器件来生成烧蚀图案，这些烧蚀图案可以被调整以改善细胞内的焦点，并动态地成形和定位焦点区域以适应不断变化的细胞结构和功能状态。2.使用激光显微手术工具，我们将分析微管（MT）阵列在活跃分裂细胞中的组装/拆卸机制。结合LC-PolScope，我们将以高的空间和时间分辨率记录MT损失和再生的星形阵列，动粒束和极间纺锤体MT的结构后果，阐明区分这些微管类的动态特性。3.我们建议开发一种成像系统，用于快速和全面分析在表达荧光标记（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。目标将是全面的光谱和偏振分析的荧光标记的蛋白质，重点是动态组织的分子聚集体细胞分裂必不可少的。4.测量荧光各向异性的仪器将应用于两个模型：（1）酵母细胞中位于芽颈的沙漏和环形结构中Septin细丝的动态组织;（2）动粒蛋白的结构-功能关系，其张力和释放的自然循环对所有真核细胞都很重要;对于后者，将使用酵母GFP标记的Ndc 80（一种普遍存在的动粒蛋白）进行初始工作。公共卫生相关性：项目叙述与以往的资助期一样，在此期间开发的显微镜仪器将在医学研究和临床环境中应用。此外，即将在该赠款下进行的细胞生物学应用项目将对我们理解细胞分裂产生广泛影响，细胞分裂是健康和患病组织中的基本细胞过程。

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.