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 莫尔纳，威廉 E. 细胞活动的复杂性需要大量酶的协调， 细胞的纳米机器及其对蛋白质和寡核苷酸的作用。蜂窝系统存储信息 以几乎永久的形式存在于细胞 DNA 中，被转录为远程蛋白质的有用信息 mRNA 中的合成。 DNA 在细胞核中的组织（位置）和运动代表了一项重要的研究 一种细胞信息流，但人们对这些细胞的精确三维运动知之甚少 细胞中的分子。因为细胞中主要生物分子的大小范围为 10 nm，在生命系统中需要对这种尺寸进行测量。相对无创的能力 直到最近，用于观察细胞行为的光学和荧光显微镜仍受到阻碍 可见光的光学衍射极限约为 200 nm。这项工作的主要目标是加强和进一步 三维（3D）光学方法，用于在前所未有的空间和动态下检查位置和动态 活细胞的时间精度。 该应用提出了当前研究的延续和扩展，以提取 3D 位置 活细胞中的单标记生物分子具有高时间分辨率和远距离的空间精度和准确度 超出光学衍射极限，低至 10-20 nm 水平。使用的关键实验工具是我们最近 开发了双螺旋点扩散函数（DH-PSF）显微镜，一种可以实现的装置 通过对传统的宽视场落射荧光或全内反射显微镜进行简单修改。我们 应用偏振传感和新的光瞳平面处理方法来扩展性能。初级 分析工具涉及统计图像处理、小波分析和压缩感知来提取隐藏的 轨迹中的信息。这项研究的目标是将DH-PSF显微镜推向最高水平 高速定位 x、y 和 z 方向的精确度和准确度，从而获得位置 细胞中的信息接近分子尺度，并将该方法应用于特定的生物 问题。 该计划有两个关键目标： 目标 1：以高收集量提取单一荧光团的方向 效率，以便将细胞中的 xyz 定位精度推至最高水平。光子效率高， 能够校正单分子偶极子定位的偏振传感 DH-PSF 显微镜设计 将建立并验证光瞳平面处理产生的误差。目标 2：应用 DH-PSF 推断相对值 活细胞中 DNA 位点动态运动的定位和变化。因为 DNA 位点的 xyz 运动 在各种基因激活条件下，在约 10 毫秒的时间尺度上，约 10-20 纳米的精度是未知的，我们 将以前所未有的水平测量单个基因座和基因座对的时间依赖性轨迹 定量。