Technical Developments in Scanning Force Microscopy for Bioimaging Applications. Studies of the Structure of E. coli RNA Polymerase

该提案描述了一个程序，以发展扫描力显微镜（SFM）作为生物学结构方法的应用。与其他显微镜相比，SFM作为生物样品结构表征的工具具有以下几个优点：1)它的分辨率与电子显微镜（EM）大致相当；Ii)成像可以在生理相关条件下进行，即在空气和液体中；iii)产生的地形图像是其他同等分辨率的显微镜难以获得的；iv)它使用的样品沉积方法比EM所需的方法要温和得多。在过去的两年里，我们的努力集中在建立扫描力显微镜作为一种直接可视化的方法，能够在高分辨率和保持其原始状态的条件下对生物结构进行成像。在此期间，我们有：i)开发了可靠的方法，在云母上沉积核酸和蛋白质/DNA复合物，并在控制湿度的空气中成像；ii)通过使用锐利碳“超级尖端”，提高了空气中生物分子的SFM空间分辨率；iii)获得了一些生物大分子在水环境和其他液体环境中的第一批图像。由于这些努力，空气中生物样本的SFM成像现在是常规和可靠的，包括我们自己的实验室在内的各种实验室现在都参与了SFM在结构生物学中的几种应用。然而，很明显，SFM的最终技术限制尚未达到，在充分实现SFM在结构生物学中的潜力之前，还需要进一步的技术发展。这些发展必须发生在三个主要领域：空间分辨率、尖端-样品相互作用的控制和生理缓冲下的成像。这个应用程序有两个部分。在A部分中，我们根据过去两年中在我们实验室获得的初步数据提出了旨在解决这些重要技术问题的实验。这项工作的具体目标是：i)为SFM开发和测试新的超锋利尖端；ii)使用非接触式操作方式；iii)设计和测试用于液体成像的新型电化学样品沉积方法。B部分描述了A部分成功技术发展的应用，即大肠杆菌RNA聚合酶DNA复合物在起始和延伸过程中的结构表征。u ~ @本提案描述了一个开发扫描力显微镜（SFM）作为结构的应用程序。K @ e Õ ' ' ' ' ' f；CG时代符号& & Arial 1 Courier 0 MS LineDraw 3 h n n E = abstract Deseree King, BIR Deseree King， BIR

9318945 Bustamante This proposal describes a program to develop the applications of Scanning Force Microscopy (SFM) as a structural method in biology. SFM offers several advantages over other microscopies as a tool for the structural characterization of biological samples because: i) It has resolution roughly comparable to the electron microscope (EM); ii) imaging can be done under physiologically relevant conditions, i.e. in air and in liquids; iii) It produces topographic images which are harder to obtain with other microscopes of equivalent resolution; and iv) It uses methods of sample deposition which are significantly gentler than those required in EM. During the last two years our efforts have been concentrated in establishing Scanning Force Microscopy as a method of direct visualization capable of imaging biological structures at high resolution and in conditions preserving their native state. In this period we have: i) Developed reliable methods of deposition of nucleic acids and protein/DNA complexes on mica and image them in air, under controlled humidity; ii) Improved the spatial resolution of SFM with biomolecules in air through the use of sharp carbon "supertips"; and iii) Obtained some of the first images of biological macromolecules in aqueous and other liquid environments. Party as, a result of these efforts, SFM imaging of biological samples in air is now routine and reliable, and various laboratories including our own are now involved in several applications of SFM to structural biology. However, it is apparent that the ultimate technical limitations of SFM have not yet been reached and that a dditional technical developments will be necessary before the potential of SFM in structural biology can be fully realized. These developments must occur in three main areas: spatial resolution, control of tip-sample interactions, and imaging under physiological buffers. This application has two parts. In part A we propose experiments designed to address each of these important technical issues based on preliminary data obtained in our laboratory during the last two years. The specific objectives of this effort are: i) Development and test of new ultra sharp tips for SFM; ii) Use of non contact modes of operation; and iii) Design and test of new electrochemical methods of sample deposition for imaging under liquids. Part B describes the application of successful technical developments of part A we the structural characterization of E. coli RNA polymerase DNA complexes during initiation and elongation. u ~ @ This proposal describes a program to develop the applications of Scanning Force Microscopy (SFM) as a struc & ? K @ E Õ ' ' ' ' ' F ; CG Times Symbol & Arial 1 Courier 0 MS LineDraw 3 h n En E = abstract Deseree King, BIR Deseree King, BIR