3D Dynamics of Cellular Information Flow

：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，以及它们在蛋白质和寡核苷酸上的工作。蜂窝系统存储信息 以几乎永久的形式存在于细胞DNA中，它被转录成有用的信息，用于远程蛋白质 mRNA的合成。DNA在细胞核中的组织（位置）和运动代表了一个重要的 这种细胞信息流，但很少有人知道这些精确的三维运动 细胞中的分子。因为细胞中的主要生物分子的大小范围是10 纳米，在生命系统中需要在这个尺寸尺度上进行测量。相对非侵入性的能力， 光学和荧光显微镜观察细胞行为一直受到阻碍，直到最近， 可见光的光学衍射极限约为200 nm。这项工作的主要目的是加强和进一步 三维（3D）光学方法，用于在前所未有的空间和 活细胞中的时间精确度。 本申请提出了当前研究的延续和扩展，以提取3D位置， 在活细胞中的单个标记的生物分子，具有高时间分辨率， 超过光学衍射极限，低至10-20 nm水平。目前使用的关键实验工具是我们最近 开发的双螺旋点扩散函数（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的简单修改。我们 应用偏振感测和新的光瞳平面处理方法来扩展性能。主 分析工具涉及统计图像处理，小波分析和压缩传感，以提取隐藏的 轨迹中的信息。本研究的目标是将DH-PSF显微镜推向最高水平 在x、y和z方向的定位精度和准确度的可能水平，从而获得位置信息。 细胞中接近分子尺度的信息，并将该方法应用于特定的生物学 问题. 两个关键目标定义了这个程序：目标1：提取具有高收集的单个荧光团的方向 效率，以便将XYZ定位精度推到细胞中的最高水平。一种光子效率高， 可校正单分子偶极定位的偏振传感DH-PSF显微镜设计 将建立并验证通过光瞳平面处理的误差。目的2：应用DH-PSF推断相对 在活细胞中DNA基因座的动态运动中的定位和变化。因为DNA位点的xyz运动 在各种基因激活条件下，在~10 ms的时间尺度上以~10-20 nm的精度未知，我们 将测量单个位点和位点对的时间依赖性轨迹， 定量

: 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.