3D Dynamics of Cellular Information Flow

蜂窝信息流的 3D 动力学

• 批准号: 8502216
• 负责人: William E Moerner
• 金额: $ 31.59万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8502216
• 关键词: Affect; Behavior; Biological; Cell Nucleus; Cells; Chromosome Structures; Chromosomes; Collection; Complex; DNA; Development; Dimensions; Disease; Enzymes; Fluorescence Microscopy; Gene Activation; Genes; Genetic Transcription; Goals; Grant; Image; Image Analysis; Knowledge; Label; Life; Location; Mammalian Cell; Measurement; Measures; Messenger RNA; Methods; Microscope; Microscopy; Modification; Molecular; Motion; Oligonucleotides; Optical Methods; Optics; Organism; Pattern; Performance; Photons; Positioning Attribute; Process; Protein Biosynthesis; Proteins; Pupil; Relative (related person); Research; Resolution; Sampling; Site; Speed; Spottings; Structure; System; Testing; Three-Dimensional Imaging; Time; Translations; Validation; Viral; Visible Radiation; Work; Yeasts; base; bioimaging; cell motility; cellular imaging; design; flexibility; fluorophore; image processing; imaging detector; improved; molecular scale; nanomachine; novel; optical imaging; particle; programs; response; single molecule; tool; two-dimensional

: GM85437-05A1 Moerner,William E. The complexity of cellular activities requires the coordination of a huge array of enzymes, the nanomachines of the cell, and their work on proteins and oligonucleotides. Cellular systems store information in a virtually permanent form in the cellular DNA, which is transcribed into useful messages for remote protein synthesis in mRNA. The organization (location) and motions of DNA in the nucleus represent one important kind of cellular information flow, yet little is known about the precise three-dimensional motions of these molecules in cells. Because the primary biomolecular players in cells are in the size range on the order of 10 nm, measurements are needed on this size scale in living systems. The relatively noninvasive capability of optical and fluorescence microscopy to observe behavior in cells has been hindered until recently by the optical diffraction limit of ~200 nm for visible light. It is a primary thrust of this work to enhance and further three-dimensional (3D) optical methods for examining locations and dynamics at unprecedented spatial and temporal precision in living cells. This application proposes continuation and expansion of current research to extract 3D positions of single labeled biomolecules in living cells with high time resolution and with spatial precision and accuracy far beyond the optical diffraction limit, down to the 10-20 nm level. The key experimental tool in use is our recently developed double-helix point spread function (DH-PSF) microscope, an apparatus that may be implemented by simple modification of a conventional wide-field epifluorescence or total-internal-reflection microscope. We apply polarization sensing and new pupil plane processing methods to extend performance. The primary analysis tools involve statistical image processing, wavelet analysis, and compressed sensing to extract hidden information in the trajectories. The goals of this research are to push the DH-PSF microscope to the highest possible levels of localization precision and accuracy in x, y, and z with high speed and thus obtain positional information in cells approaching the molecular scale, and to apply the approach to a specific biological problem. Two key aims define this program: Aim 1: Extract orientation of single fluorophores with high collection efficiency in order to push xyz localization accuracy to the highest levels in cells. A photon-efficient, polarization-sensing DH-PSF microscope design capable of correcting single-molecule dipole localization errors by pupil plane processing will be built and validated. Aim 2: Apply the DH-PSF to infer relative positioning and changes in dynamical motions of DNA loci in living cells. Because the xyz motions of DNA loci under various gene activation conditions are not known on the ~10 ms time scale with ~10-20 nm precision, we will measure the time-dependent trajectories of single loci and pairs of loci with unprecedented levels of quantitation.

：GM85437-05A1 Moerner，William E. 细胞活性的复杂性需要大量酶的协调， 细胞的纳米机器，以及它们在蛋白质和寡核苷酸上的工作。蜂窝系统存储信息 在细胞DNA中的几乎永久形式中，该形式被转录为远程蛋白质的有用消息 mRNA中的合成。核中DNA的组织（位置）和运动代表一个重要的 一种细胞信息流，但对这些的精确三维运动知之甚少 细胞中的分子。因为细胞中的主要生物分子玩家的大小范围为10 NM，在生活系统中需要在此尺寸规模上进行测量。相对无创的能力 直到最近，光学和荧光显微镜观察细胞中的行为一直受到阻碍 可见光的光学衍射极限为〜200 nm。这是这项工作的主要作用，以增强和进一步 三维（3D）光学方法，用于检查前所未有的空间和动力学 活细胞的时间精度。 该申请提出了当前研究的延续和扩展，以提取3D位置 具有较高时间且具有空间精度和精度的活细胞中的单个标记的生物分子 超出光学衍射极限，直至10-20 nm水平。使用的关键实验工具是我们最近 开发的双螺旋点扩展功能（DH-PSF）显微镜，可以实现的一种设备 通过简单地修改常规的宽田表荧光或总内部反射显微镜。我们 应用偏振感和新的瞳孔处理方法来扩展性能。主要 分析工具涉及统计图像处理，小波分析和压缩感应以提取隐藏 轨迹中的信息。这项研究的目标是将DH-PSF显微镜推向最高 X，Y和Z中可能的定位精度和高速的精度水平，从而获得位置 接近分子尺度的细胞中的信息，并将方法应用于特定的生物学 问题。 两个关键目的定义了以下程序：目标1：提取高收集的单个荧光团的方向 效率以将XYZ定位精度推向细胞中最高水平。光子效率， 极化感应DH-PSF显微镜设计，能够校正单分子偶极子定位 将建立和验证学生平面处理的错误。 AIM 2：将DH-PSF应用于推断亲戚 活细胞中DNA基因座的动态运动的定位和变化。因为DNA基因座的XYZ运动 在各种基因激活条件下，在〜10 ms的时间尺度上以〜10-20 nm的精度不知道，我们 将测量单个基因座和对基因座的时间依赖性轨迹，并具有前所未有的水平 定量。