3D Dynamics of Cellular Information Flow

点击翻译按钮获取中文摘要

: 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.