Advanced Optical Methods in Cell Biology

项目概述/摘要：在我们开发超灵敏和高分辨率的液晶偏光显微镜（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. 我们提出了一种基于固态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来产生烧蚀模式，这些模式可以调谐以改善细胞内的焦点，并动态地塑造和定位焦点区域，以适应不断变化的细胞结构和功能状态。2. 利用激光显微手术工具，我们将分析活跃分裂细胞中微管（MT）阵列的组装/拆卸机制。结合LC-PolScope，我们将以高空间和时间分辨率记录在星状阵列、着丝点束和极间纺轴MT中MT损失和再生的结构后果，揭示区分这些微管类别的动态特性。3. 我们建议开发一种成像系统，用于快速和全面地分析在表达荧光标记（例如GFP标记）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极化分析，重点是细胞分裂所必需的分子聚集体的动态组织。4. 测量荧光各向异性的仪器将应用于两个模型：(1)酵母细胞中沙漏中间隔蛋白丝的动态组织和位于芽颈的环状结构；(2)着丝点蛋白的结构-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，最初的工作将使用酵母GFP标记的Ndc80，一种普遍存在的着丝点蛋白。与公共卫生相关：项目叙述与以前的资助期一样，在此期间开发的显微镜仪器将在医学研究和临床环境中得到应用。此外，这项资助下即将开展的细胞生物学应用项目将对我们对细胞分裂的理解产生广泛的影响，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.