结构生物学：STM and AFM

1、一.STM原理简介 1982年，国际商业机器公司苏黎世实验室的G.Binnig和HeinrichRohrer及其同事们共同研制成功了世界上第一台新型的表面分析仪器扫描隧道显微镜(Scanning Tunneling Microscope，简称STM)。STM的出现，使人类第一次能够实时地观察单观察单个原子在物质表面的排列状态个原子在物质表面的排列状态，研究与表面电子电子行为有关的物理和化学性质,1986年宾尼和罗雷尔因此获得诺贝尔物理学奖。 STM与两位发明人 STM的工作原理的工作原理 STM是利用量子隧道效应工作的。若以金属针尖为一电极，被测固体样品为另一电极，当他们之间的距离小到1nm左

2、右时，就会出现隧道效应，电子从一个电极穿过空间势垒到达另一电极形成电流。 且其中Vb：偏置电压； A：常数，约等于1， 1/2：平均功函数， S：距离。 样品要有导电性样品要有导电性隧道电流定量关系：隧道电流定量关系： I Vbexp(-A 1/2 S) S与样品表面特性相关的参数； Vb探针跟样品之间的偏压（电压差） 在针尖逼近样品表面时（1纳米）； S有0.1纳米的变化； I即有数量级的变化 1针尖, 2样品 =1/2 1+1/2 2 STM的意义 STM是继高分辨透射电子显微镜，场离子显微镜之后，第三种在原子尺度观察物质表面结构的显微镜，其分辨率在水平方向可达0.1nm，垂直方向可达0.

3、01nm，它的出现标志着纳米技术研究的一个最重大的转折，这是因为STM具有原子和纳米尺度的分析和加工的能力。 在物理学和化学领域，可用于研究原子之间的微小结合能，制造人造分子； 在生物学领域，可用于研究生物细胞和染色体内的单个蛋白质和DNA分子的结构，进行分子切割和组装手术； 在材料学领域，可以用于分析材料的晶格和原子结构，考察晶体中原子尺度上的缺陷； 在微电子领域，则可以用于加工小至原子尺度的新型量子器件。 中国第一批扫描隧道显微镜诞生中国第一批扫描隧道显微镜诞生在扫描隧道显微镜发明者获得1986年诺贝尔奖的同时，STM的神奇魅力也深深打动了一批中国学者的心。在美国加州理工学院中科院化学所白

4、春礼博士正从事着STM的研制工作；北京中科院电子显微镜实验室姚骏恩研究员从电子显微镜向第三代的扫描显微镜的过渡北大物理系的杨威生教授，希望建立起高真空下的STM，以观察半导体，金属表面的原子结构，但第一步得先把常压下的STM试制出来吧。中科院上海原子核所的李民乾研究员思考着怎么从依赖庞大设备的应用核物理研究转向同样有价值的“小科学”？STM是一个理想方向！于是他决心放弃熟悉的、自己亲自发展起来的多项核分析技术，转向扫描隧道显微学及其应用领域。他与胡均、顾敏明和徐耀良等一起详细研究了STM的各种设计，觉得STM的特点是多参数的数据收集和处理，这正是核物理实验中最熟悉的方式，国产化的STM完全有可

5、能在短期内研制成功。在80年代末的报纸上先后报道了上述四个单位研制成功STM的消息。以白春礼领衔，中国第一批的扫描隧道显微镜诞生了。在当时尚无成熟商品化STM的情况下，自己研制无疑在启动我国的纳米科技研究方面起了重要作用.。恒高模式 隧道电流与针尖样品间距S成负指数关系。对于间距的变化非常敏感。因此，当针尖在被测样品表面做平面扫描时，即使表面仅有原子尺度的起伏，也会导致隧道电流的非常显著的、甚至接近数量级的变化。这样就可以通过测量电流的变化来反应表面上原子尺度的起伏，这就是STM的基本工作原理，这种运行模式称为恒高模式（保持针尖高度恒定）。 恒流模式 STM恒流模式:针尖扫描过程中，通过电子反

6、馈回路保持隧道电流不变。为维持恒定的电流，针尖随样品表面的起伏上下移动，从而记录下针尖上下运动的轨迹，即可给出样品表面的形貌。 恒流模式是STM常用的工作模式，而恒高模式仅适于对表面起伏不大的样品进行成像。当样品表面起伏较大时，由于针尖离样品表面非常近，采用恒高模式扫描容易造成针尖与样品表面相撞，导致针尖与样品表面的破坏。 “足球分子足球分子” C60的庐山真面貌的庐山真面貌 在千变万化的微观世界里，存在着一种与足球的形态十分相似的分子，人们称其为“C60”，又称“富勒烯”、“巴基球”。这种由60个碳原子组成的“足球分子”是英国化学家克鲁托（H.W. Kroto）、美国化学家斯莫利(R.E.

7、Smalley)与柯尔(R.F. Curl)发现的。1996年上述三位科学家因为发现了这种碳元素的第三种晶体存在形式而分享了当年的诺贝尔化学奖。C60分子中的所有碳原子都是通过共价键构成的，形成具有12个五边形和20个六边形的笼状结构。其中每个五边形的五条边是碳碳单键，五边形的顶点与周围的五边形的顶点之间是碳碳双键。中国科技大学侯建国教授领导的课题组将C60分子组装在单层分子膜的表面，隔绝了金属衬底的影响，在零下268度下，将分子热运动冻结，利用扫描隧道显微镜（STM）在国际上首次“拍下”了能够分辨碳碳单键和双键的分子图象。C60分子的结构模型图与STM图象 小分子的操纵小分子的操纵 美国加州

8、IBM研究实验室的访问学者泽彭菲尔德（P.Zeppenfield）利用STM操纵小分子，将28个一氧化碳分子在铂金的表面上排布成了世界上最小的“分子人” ）CO(一氧化碳)分子小人（身高5纳米） 二.STM应用于结构生物学 1. STM观察样品的三维结构 2. STM适用不同的探测环境 3. STM观测范围可变，不同层次生命结构 4.STM制样简单，量少，成本低1.核酸的STM研究 中国科学院上海原子核所应用自己研制成功的STM与上海细胞生物学所及前苏联科学院分子生物学所合作获得的一种新的DNA构型一一平行双链DNA(PDNA)的STM图像，直观地显示了PDNA的结构特征：右手螺旋及链的等距间

9、隔(从照片上则相应地可看到右手螺旋和等距间隔的条纹)。 （1）平行双链DNA(PDNA)的STM图像 （2）大气中的）大气中的DNA和和RNAScience. 1989 Jan 20;243(4889):370-2. Links Direct observation of native DNA structures with the scanning tunneling microscope. Beebe TP Jr, Wilson TE, Ogletree DF, Katz JE, Balhorn R, Salmeron MB, Siekhaus WJ. Lawrence Livermore

10、 National Laboratory, Department of Chemistry, Livermore, CA 94550. Uncoated double-stranded DNA dissolved in a salt solution was deposited on graphite and imaged in air with the scanning tunneling microscope (STM). The resolution was such that the major and minor grooves could be distinguished. The

11、 pitch of the helix varied between 27 and 63 angstroms in the images obtained. Thus the STM can be useful for structural studies of a variety of uncoated and isolated biomolecules.Science. 1991 Feb 8;251(4994):640-2. Links: Graphite: a mimic for DNA and other biomolecules in scanning tunneling micro

12、scope studies. Clemmer CR, Beebe TP Jr. Department of Chemistry, University of Utah, Salt Lake City 84112. Highly ordered pyrolytic graphite (HOPG) is the substrate often used in scanning tunneling microscope (STM) studies of biomolecules such as DNA. All of the images presented in this article are

13、of freshly cleaved HOPG surfaces upon which no deposition has occurred. These images illustrate features previously thought to be due to biological molecules, such as periodicity and meandering of molecules over steps. These features can no longer be used to distinguish real molecules from features

14、of the native substrate. The feasibility of the continued use of HOPG as a substrate for biological STM studies is discussed. PMID: 1992517 PubMed - indexed for MEDLINE（3）Z-DNA,A-RNA,单链单链DNANature 339, 484-486 (8 June 1989) | Scanning tunnelling microscopy of Z-DNAPatricia G. Arscott*, Gil Lee, Vict

15、or A. Bloomfield* & D. Fennell Evans* Department of Biochemistry, 1479 Gortner Avenue, University of Minnesota, St Paul, Minnesota 55108, USA Department of Chemical Engineering and Materials Science, and Interfacial Sciences Center, Shepherd Laboratory, 100 Union Street S.E., University of Minne

16、sota, Minneapolis, Minnesota 55455, USAAbstractSCANNING tunnelling microscopy (STM) has been used to map the surface topography of inorganic materials at the atomic level, and is potentially one of the most powerful techniques for probing biomolecular structure. Recent STM studies of calf thymus DNA

17、 and poly(rA) poly(rU) have shown that the helical pitch and periodic alternation of major and minor grooves can be visualized and reliably measured. Here we present the first STM images of poly(dG-me5d) poly(dG-me5dC) in the Z-form. Both the general appearance of the fibres and measurements of heli

18、cal parameters are in good agreement with models derived from X-ray diffraction.Science. 1989 Mar 31;243(4899):1708-11. Links Scanning tunneling microscopy of uncoated recA-DNA complexes. Amrein M, Drr R, Stasiak A, Gross H, Travaglini G. Institute of Cell Biology, Swiss Federal Institute of Technol

19、ogy, Zurich, Switzerland. Uncoated recA-DNA complexes were imaged with the scanning tunneling microscope (STM). The images, which reveal the right-handed helical structure of the complexes with subunits clearly resolved, are comparable in quality to STM images of metal-coated specimens. Possible con

20、duction mechanisms that allow STM imaging of biological macromolecules are discussed. PMID: 2928803 PubMed - indexed for MEDLINENature. 1990 Jul 19;346(6281):294-6. LinksAtomic-scale imaging of DNA using scanning tunnelling microscopy.Driscoll RJ, Youngquist MG, Baldeschwieler JD.Division of Chemist

21、ry and Chemical Engineering, California Institute of Technology, Pasadena 91125.The scanning tunnelling microscope (STM) has been used to visualize DNA under water, under oil and in air. Images of single-stranded DNA have shown that submolecular resolution is possible. Here we describe atomic-resolu

22、tion imaging of duplex DNA. Topographic STM images of uncoated duplex DNA on a graphite substrate obtained in ultra-high vacuum are presented that show double-helical structure, base pairs, and atomic-scale substructure. Experimental STM profiles show excellent correlation with atomic contours of th

23、e van der Waals surface of A-form DNA derived from X-ray crystallography. A comparison of variations in the barrier to quantum mechanical tunnelling (barrier-height) with atomic-scale topography shows correlation over the phosphate-sugar backbone but anticorrelation over the base pairs. This relatio

24、nship may be due to the different chemical characteristics of parts of the molecule. Further investigation of this phenomenon should lead to a better understanding of the physics of imaging adsorbates with the STM and may prove useful in sequencing DNA. The improved resolution compared with previous

25、ly published STM images of DNA may be attributable to ultra-high vacuum, high data-pixel density, slow scan rate, a fortuitously clean and sharp tip and/or a relatively dilute and extremely clean sample solution. This work demonstrates the potential of the STM for characterization of large biomolecu

26、lar structures, but additional development will be required to make such high resolution imaging of DNA and other large molecules routine.（4）DNA与蛋白质复合物 一切生命物质中的DNA的复制过程每时每刻都在进行着，但人们从未直观见过。图3.6.2a是中科院原子核所和生化所合作，利用STM拍摄到的表征DNA复制过程中一瞬间的照片。图的中央是DNA聚合酶，左下角为DNA双链分子，其相应的结构模型图见图3.6.2b。DNA复制的瞬间图像 1: Science.

27、 1988 Oct 14;242(4876):209-16. LinksScanning tunneling microscopy and atomic force microscopy: application to biology and technology.Hansma PK, Elings VB, Marti O, Bracker CE.Department of Physics, University of California, Santa Barbara 93106.The scanning tunneling microscope (STM) and the atomic f

28、orce microscope (AFM) are scanning probe microscopes capable of resolving surface detail down to the atomic level. The potential of these microscopes for revealing subtle details of structure is illustrated by atomic resolution images including graphite, an organic conductor, an insulating layered c

29、ompound, and individual adsorbed oxygen atoms on a semiconductor. Application of the STM for imaging biological materials directly has been hampered by the poor electron conductivity of most biological samples. The use of thin conductive metal coatings and replicas has made it possible to image some

30、 biological samples, as indicated by recently obtained images of a recA-DNA complex, a phospholipid bilayer, and an enzyme crystal. The potential of the AFM, which does not require a conductive sample, is shown with molecular resolution images of a nonconducting organic monolayer and an amino acid c

31、rystal that reveals individual methyl groups on the ends of the amino acids. Applications of these new microscopes to technology are demonstrated with images of an optical disk stamper, a diffraction grating, a thin-film magnetic recording head, and a diamond cutting tool. The STM has even been used

32、 to improve the quality of diffraction gratings and magnetic recording heads.PMID: 3051380 PubMed - indexed for MEDLINEScience. 1988 Apr 22;240(4851):514-6. Links Scanning tunneling microscopy of recA-DNA complexes coated with a conducting film. Amrein M, Stasiak A, Gross H, Stoll E, Travaglini G. I

33、nstitute for Cell Biology, Swiss Federal Institute of Technology ETH Hnggerberg, Zurich, Switzerland. A link between scanning tunneling microscopy (STM) and conventional transmission electron microscopy has been established for biological material by applying STM on freeze-dried recA-DNA complexes c

34、oated with a conducting film. The topography of the complexes observed by means of STM revealed a right-handed single helix composed of about six recA monomers per helical turn. PMID: 3358130 PubMed - indexed for MEDLINEScience. 1988 Oct 14;242(4876):209-16. LinksScanning tunneling microscopy and at

35、omic force microscopy: application to biology and technology.Hansma PK, Elings VB, Marti O, Bracker CE.Department of Physics, University of California, Santa Barbara 93106.The scanning tunneling microscope (STM) and the atomic force microscope (AFM) are scanning probe microscopes capable of resolvin

36、g surface detail down to the atomic level. The potential of these microscopes for revealing subtle details of structure is illustrated by atomic resolution images including graphite, an organic conductor, an insulating layered compound, and individual adsorbed oxygen atoms on a semiconductor. Applic

37、ation of the STM for imaging biological materials directly has been hampered by the poor electron conductivity of most biological samples. The use of thin conductive metal coatings and replicas has made it possible to image some biological samples, as indicated by recently obtained images of a recA-

38、DNA complex, a phospholipid bilayer, and an enzyme crystal. The potential of the AFM, which does not require a conductive sample, is shown with molecular resolution images of a nonconducting organic monolayer and an amino acid crystal that reveals individual methyl groups on the ends of the amino ac

39、ids. Applications of these new microscopes to technology are demonstrated with images of an optical disk stamper, a diffraction grating, a thin-film magnetic recording head, and a diamond cutting tool. The STM has even been used to improve the quality of diffraction gratings and magnetic recording h

40、eads.PMID: 3051380 PubMed - indexed for MEDLINE（5）单个）单个DNA分子的操纵分子的操纵 在单原子和小分子操纵方面不断取得进展的同时，对最重要的生物大分子DNA分子的操纵研究也开展了。这是中国科学院上海原子核所“单分子探测和操纵”实验室自1990年以来一贯追求的目标。国际纳米科技重要刊物“纳米通讯”（Nano Letters）在2003年1月号上发表了它的首例封面故事：对DNA分子链通过单分子纳米操纵，构成了三个纳米尺度的字“DNA”，它是用DNA分子片段构建的字（纳米图案），其尺度为200300纳米。这个首创的成果是中国科学院上海原子核研究所“

41、单分子探测和操纵”实验室和上海交通大学Bio-X中心为主与德国萨莱大学(Saarland Univ)的科学家合作完成的。单个DNA分子的操纵打开了应用的大门，应用领域涉及纳米电子学、纳米生物学中的许多方面，例如DNA测序的有序化，基因突变的单分子诊断和蛋白质的纳米芯片等。2.蛋白质的STM研究功能蛋白细胞色素C2。蛋白质的STM研究（红素氧还蛋白分子）X-ray crystallographic structure of Clostridium pasteurianum rubredoxinConstant-current STMimage (35=35 nm2) of rubredoxinm

42、oleculeson gold (111) surface. Bias 900 mV on the tip, tunnelling current 200 pA,scan rate 267 nm/s, z-range 00.6 nm.At thismagnification, a sub-molecularfeature is visible in the outlined molecules.A 3D view (57=57 nm2) of rubredoxin molecules on gold (111)surface. A contrast enhancement is clearly

43、 visible over the highlightedmolecules.High-magnification STM image (12=12 nm2) of single rubredoxinmolecule. Bias 900 mV, tunnelling current 200 pA, scan rate 90 nm/s,z-range 00.6 nm.AFM的工作原理的工作原理 AFM（Atomic 的基本原理与STM类似，在AFM中，使用对微弱力非常敏感的弹性悬臂上的针尖对样品表面作光栅式扫描。当针尖和样品表面的距离非常接近时，针尖尖端的原子与样品表面的原子之间存在极微弱的作用

44、力（10-1210-6N），此时，微悬臂就会发生微小的弹性形变。针尖与样品之间的力F与微悬臂的形变 之间遵循虎克定律：F=-k*x ，其中，k为微悬臂的力常数。所以，只要测出微悬臂形变量的大小，就可以获得针尖与样品之间作用力的大小。 “恒力”模式 针尖与样品之间的作用力与距离有强烈的依赖关系，所以在扫描过程中利用反馈回路保持针尖与样品之间的作用力恒定，即保持为悬臂的形变量不变，针尖就会随样品表面的起伏上下移动，记录针尖上下运动的轨迹即可得到样品表面形貌的信息。这种工作模式被称为“恒力”模式（Constant Force Mode），是使用最广泛的扫描方式。 “恒高”模式 AFM的图像也可以使用

45、“恒高”模式（Constant Height Mode）来获得，也就是在X，Y扫描过程中，不使用反馈回路，保持针尖与样品之间的距离恒定，通过测量微悬臂Z方向的形变量来成像。这种方式不使用反馈回路，可以采用更高的扫描速度，通常在观察原子、分子像时用得比较多，而对于表面起伏比较大的样品不适用。 AFM的优点： 原子级水平 各种条件下 样品制备简单 真的表面结构 实时动态快速过程 小尺寸样品的加工实验条件 基底选择 探针 制样AFM在结构生物学中的应用 生物大分子（130nm的DNA的片段） 细胞、细胞膜、人工质子体 病毒、细菌、微生物。抗原、抗体 生物表面事件的实时原位监测（动态）10-100S（细胞的分裂和RNA翻译）: J Struct Biol. 1990 Oct-Dec;105(1-3):54-61. Links Imaging cells with the atomic force microscope. Butt HJ, Wolff EK, Gould SA, Dixon Northern B, Peterson CM, Hansma PK. Department of Physics, University of California