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

9318945布斯塔曼特本提案描述了一项开发扫描力显微镜在生物学中作为一种结构方法的应用的计划。作为生物样品的结构表征工具，SFM与其他显微镜相比具有几个优势，因为：i)它的分辨率与电子显微镜(EM)大致相当；ii)成像可以在与生理相关的条件下进行，即在空气和液体中；iii)它产生的地形图像比其他同等分辨率的显微镜更难获得；以及iv)它使用的样品沉积方法比EM所要求的方法温和得多。在过去的两年里，我们一直致力于建立扫描力显微镜作为一种直接可视化的方法，能够以高分辨率和在保持其原始状态的条件下对生物结构进行成像。在这一时期，我们已经：i)开发了可靠的方法，将核酸和蛋白质/DNA复合体沉积在云母上，并在受控湿度下在空气中成像；ii)通过使用锋利的碳“超级尖端”提高了SFM与空气中生物分子的空间分辨率；以及iii)获得了水环境和其他液体环境中生物大分子的一些第一批图像。作为这些努力的结果，空气中生物样品的SFM成像现在是常规和可靠的，包括我们自己在内的各种实验室现在参与了SFM在结构生物学中的几个应用。然而，很明显，SFM的最终技术极限还没有达到，在SFM在结构生物学中的潜力得以充分发挥之前，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-样本相互作用的控制，以及生理缓冲下的成像。这个应用程序由两部分组成。在A部分中，我们根据我们实验室在过去两年中获得的初步数据，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)开发和测试用于SFM的新的超锐尖；ii)使用非接触式操作模式；以及iii)设计和测试用于液体下成像的新的电化学样品沉积方法。B部分描述了A部分的成功技术开发的应用，我们描述了E.Coli RNA聚合酶DNA复合体在起始和延伸过程中的结构特征。U~@这份提案描述了一项开发扫描力显微镜(SFM)作为结构应用的计划。K@E？‘F；CG时间符号和宋体1信使0 MS线条绘制3小时n en E=抽象沙漠王，BIR沙漠王，BIR沙漠王

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