3D Dynamics of Cellular Information Flow

书名：GM85437-05A1 Moerner，William E. 细胞活动的复杂性需要大量酶的协调， 细胞的纳米机器，以及他们在蛋白质和寡核苷酸方面的工作。蜂窝系统存储信息 在细胞DNA中以一种几乎永久的形式存在，它被转录成对远程蛋白质有用的信息 信使核糖核酸合成。DNA在核中的组织(位置)和运动代表了一个重要的 一种细胞信息流，但对这些细胞的精确三维运动知之甚少 细胞中的分子。因为细胞中的主要生物分子分子的大小约为10 在生命系统中，这种规模的测量是需要的。相对非侵入性的能力 直到最近，用于观察细胞行为的光学和荧光显微镜一直受到 可见光的光学衍射极限为~200 nm。这项工作的主旨是加强和进一步 三维(3D)光学方法，用于在前所未有的空间和 活细胞的时间精确度。 这项应用建议继续和扩展当前的研究，以提取三维位置 活细胞中的单标记生物分子具有高时间分辨率和高空间精度 超过光学衍射极限，下至10-20 nm级别。正在使用的关键实验工具是我们最近的 研制的双螺旋点扩散函数显微镜，一种可能实现的装置 通过对传统的广视场荧光显微镜或全内反射显微镜进行简单的改装。我们 应用偏振传感和新的光瞳平面处理方法来扩展性能。初级阶段 分析工具包括统计图像处理、小波分析和压缩传感以提取隐藏信息 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 以高速在x、y和z方向上的定位精度和准确度的可能水平，从而获得 细胞中的信息接近分子尺度，并将这种方法应用于特定的生物 有问题。 这项计划的两个关键目标是：目标1：高收集率的单一荧光团的提取方向 效率，以便将XYZ定位精度推向细胞内的最高水平。一种光子效率高的， 可校正单分子偶极子局域化的偏振传感DH-PSF显微镜设计 将建立并验证光瞳平面处理的误差。目的2：应用DH-PSF推断亲缘关系 DNA基因座在活细胞中的定位和动态运动变化。因为DNA基因座的XYZ运动 在不同的基因激活条件下，在~10ms的时间尺度上未知，精度~10-20 nm，我们 将以前所未有的水平测量单个基因座和成对基因座的随时间变化的轨迹 量化。

: 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.