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

9318945 Bustamante该提案描述了一个程序，以开发扫描力显微镜（SFM）作为生物学中的结构方法的应用。作为生物样品结构表征的工具，SFM提供了优于其他显微镜的几个优点，因为：i）它具有与电子显微镜（EM）大致相当的分辨率; ii）成像可以在生理相关条件下进行，即在空气和液体中; iii）它产生的地形图像难以用同等分辨率的其他显微镜获得;和iv）它使用比EM中所需的那些显著更温和的样品沉积方法。 在过去的两年中，我们的努力一直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自然状态的条件下对生物结构进行成像。在此期间，我们有：㈠开发了将核酸和蛋白质/DNA复合物沉积在云母上并在空气中在受控湿度下对其成像的可靠方法; ㈡通过使用尖锐的碳“超级尖端”，提高了空气中生物分子的SFM的空间分辨率; ㈢获得了水和其他液体环境中生物大分子的一些首批图像。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个应用。 然而，很明显，SFM的最终技术限制还没有达到，并且在SFM在结构生物学中的潜力可以完全实现之前，需要进行快速的技术开发。这些发展必须发生在三个主要领域：空间分辨率，控制尖端样品相互作用，和生理缓冲下的成像。 此应用程序有两个部分。在A部分，我们提出了旨在解决这些重要的技术问题的基础上，在我们的实验室在过去两年中获得的初步数据的实验。这项工作的具体目标是：i）开发和测试用于SFM的新的超锋利尖端; ii）使用非接触操作模式; iii）设计和测试用于液体下成像的样品沉积的新电化学方法。B部分描述了A部分的成功技术开发的应用，我们对E.大肠杆菌RNA聚合酶DNA复合物的起始和延伸过程中。 u ~ 本提案描述了一个开发扫描力显微镜（SFM）应用程序的计划， & ? K @ E Õ ' ' ' ' ' F ; CG Times Symbol Arial 1 CXCR0 MS LineDraw 3 H n恩E = 摘要 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