結(jié)構(gòu)生物學：STM and AFM

1、一.STM原理簡介 1982年，國際商業(yè)機器公司蘇黎世實驗室的G.Binnig和HeinrichRohrer及其同事們共同研制成功了世界上第一臺新型的表面分析儀器掃描隧道顯微鏡(Scanning Tunneling Microscope，簡稱STM)。STM的出現(xiàn)，使人類第一次能夠?qū)崟r地觀察單觀察單個原子在物質(zhì)表面的排列狀態(tài)個原子在物質(zhì)表面的排列狀態(tài)，研究與表面電子電子行為有關的物理和化學性質(zhì),1986年賓尼和羅雷爾因此獲得諾貝爾物理學獎。 STM與兩位發(fā)明人 STM的工作原理的工作原理 STM是利用量子隧道效應工作的。若以金屬針尖為一電極，被測固體樣品為另一電極，當他們之間的距離小到1nm左

2、右時，就會出現(xiàn)隧道效應，電子從一個電極穿過空間勢壘到達另一電極形成電流。 且其中Vb：偏置電壓； A：常數(shù)，約等于1， 1/2：平均功函數(shù)， S：距離。 樣品要有導電性樣品要有導電性隧道電流定量關系：隧道電流定量關系： I Vbexp(-A 1/2 S) S與樣品表面特性相關的參數(shù)； Vb探針跟樣品之間的偏壓（電壓差） 在針尖逼近樣品表面時（1納米）； S有0.1納米的變化； I即有數(shù)量級的變化 1針尖, 2樣品 =1/2 1+1/2 2 STM的意義 STM是繼高分辨透射電子顯微鏡，場離子顯微鏡之后，第三種在原子尺度觀察物質(zhì)表面結(jié)構(gòu)的顯微鏡，其分辨率在水平方向可達0.1nm，垂直方向可達0.

3、01nm，它的出現(xiàn)標志著納米技術研究的一個最重大的轉(zhuǎn)折，這是因為STM具有原子和納米尺度的分析和加工的能力。 在物理學和化學領域，可用于研究原子之間的微小結(jié)合能，制造人造分子； 在生物學領域，可用于研究生物細胞和染色體內(nèi)的單個蛋白質(zhì)和DNA分子的結(jié)構(gòu)，進行分子切割和組裝手術； 在材料學領域，可以用于分析材料的晶格和原子結(jié)構(gòu)，考察晶體中原子尺度上的缺陷； 在微電子領域，則可以用于加工小至原子尺度的新型量子器件。 中國第一批掃描隧道顯微鏡誕生中國第一批掃描隧道顯微鏡誕生在掃描隧道顯微鏡發(fā)明者獲得1986年諾貝爾獎的同時，STM的神奇魅力也深深打動了一批中國學者的心。在美國加州理工學院中科院化學所白

4、春禮博士正從事著STM的研制工作；北京中科院電子顯微鏡實驗室姚駿恩研究員從電子顯微鏡向第三代的掃描顯微鏡的過渡北大物理系的楊威生教授，希望建立起高真空下的STM，以觀察半導體，金屬表面的原子結(jié)構(gòu)，但第一步得先把常壓下的STM試制出來吧。中科院上海原子核所的李民乾研究員思考著怎么從依賴龐大設備的應用核物理研究轉(zhuǎn)向同樣有價值的“小科學”？STM是一個理想方向！于是他決心放棄熟悉的、自己親自發(fā)展起來的多項核分析技術，轉(zhuǎn)向掃描隧道顯微學及其應用領域。他與胡均、顧敏明和徐耀良等一起詳細研究了STM的各種設計，覺得STM的特點是多參數(shù)的數(shù)據(jù)收集和處理，這正是核物理實驗中最熟悉的方式，國產(chǎn)化的STM完全有可

5、能在短期內(nèi)研制成功。在80年代末的報紙上先后報道了上述四個單位研制成功STM的消息。以白春禮領銜，中國第一批的掃描隧道顯微鏡誕生了。在當時尚無成熟商品化STM的情況下，自己研制無疑在啟動我國的納米科技研究方面起了重要作用.。恒高模式 隧道電流與針尖樣品間距S成負指數(shù)關系。對于間距的變化非常敏感。因此，當針尖在被測樣品表面做平面掃描時，即使表面僅有原子尺度的起伏，也會導致隧道電流的非常顯著的、甚至接近數(shù)量級的變化。這樣就可以通過測量電流的變化來反應表面上原子尺度的起伏，這就是STM的基本工作原理，這種運行模式稱為恒高模式（保持針尖高度恒定）。 恒流模式 STM恒流模式:針尖掃描過程中，通過電子反

6、饋回路保持隧道電流不變。為維持恒定的電流，針尖隨樣品表面的起伏上下移動，從而記錄下針尖上下運動的軌跡，即可給出樣品表面的形貌。 恒流模式是STM常用的工作模式，而恒高模式僅適于對表面起伏不大的樣品進行成像。當樣品表面起伏較大時，由于針尖離樣品表面非常近，采用恒高模式掃描容易造成針尖與樣品表面相撞，導致針尖與樣品表面的破壞。 “足球分子足球分子” C60的廬山真面貌的廬山真面貌 在千變?nèi)f化的微觀世界里，存在著一種與足球的形態(tài)十分相似的分子，人們稱其為“C60”，又稱“富勒烯”、“巴基球”。這種由60個碳原子組成的“足球分子”是英國化學家克魯托（H.W. Kroto）、美國化學家斯莫利(R.E.

7、Smalley)與柯爾(R.F. Curl)發(fā)現(xiàn)的。1996年上述三位科學家因為發(fā)現(xiàn)了這種碳元素的第三種晶體存在形式而分享了當年的諾貝爾化學獎。C60分子中的所有碳原子都是通過共價鍵構(gòu)成的，形成具有12個五邊形和20個六邊形的籠狀結(jié)構(gòu)。其中每個五邊形的五條邊是碳碳單鍵，五邊形的頂點與周圍的五邊形的頂點之間是碳碳雙鍵。中國科技大學侯建國教授領導的課題組將C60分子組裝在單層分子膜的表面，隔絕了金屬襯底的影響，在零下268度下，將分子熱運動凍結(jié)，利用掃描隧道顯微鏡（STM）在國際上首次“拍下”了能夠分辨碳碳單鍵和雙鍵的分子圖象。C60分子的結(jié)構(gòu)模型圖與STM圖象 小分子的操縱小分子的操縱 美國加州

8、IBM研究實驗室的訪問學者澤彭菲爾德（P.Zeppenfield）利用STM操縱小分子，將28個一氧化碳分子在鉑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納米） 二.STM應用于結(jié)構(gòu)生物學 1. STM觀察樣品的三維結(jié)構(gòu) 2. STM適用不同的探測環(huán)境 3. STM觀測范圍可變，不同層次生命結(jié)構(gòu) 4.STM制樣簡單，量少，成本低1.核酸的STM研究 中國科學院上海原子核所應用自己研制成功的STM與上海細胞生物學所及前蘇聯(lián)科學院分子生物學所合作獲得的一種新的DNA構(gòu)型一一平行雙鏈DNA(PDNA)的STM圖像，直觀地顯示了PDNA的結(jié)構(gòu)特征：右手螺旋及鏈的等距間

9、隔(從照片上則相應地可看到右手螺旋和等距間隔的條紋)。 （1）平行雙鏈DNA(PDNA)的STM圖像 （2）大氣中的）大氣中的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,單鏈單鏈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與蛋白質(zhì)復合物 一切生命物質(zhì)中的DNA的復制過程每時每刻都在進行著，但人們從未直觀見過。圖3.6.2a是中科院原子核所和生化所合作，利用STM拍攝到的表征DNA復制過程中一瞬間的照片。圖的中央是DNA聚合酶，左下角為DNA雙鏈分子，其相應的結(jié)構(gòu)模型圖見圖3.6.2b。DNA復制的瞬間圖像 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）單個）單個DNA分子的操縱分子的操縱 在單原子和小分子操縱方面不斷取得進展的同時，對最重要的生物大分子DNA分子的操縱研究也開展了。這是中國科學院上海原子核所“單分子探測和操縱”實驗室自1990年以來一貫追求的目標。國際納米科技重要刊物“納米通訊”（Nano Letters）在2003年1月號上發(fā)表了它的首例封面故事：對DNA分子鏈通過單分子納米操縱，構(gòu)成了三個納米尺度的字“DNA”，它是用DNA分子片段構(gòu)建的字（納米圖案），其尺度為200300納米。這個首創(chuàng)的成果是中國科學院上海原子核研究所“

41、單分子探測和操縱”實驗室和上海交通大學Bio-X中心為主與德國薩萊大學(Saarland Univ)的科學家合作完成的。單個DNA分子的操縱打開了應用的大門，應用領域涉及納米電子學、納米生物學中的許多方面，例如DNA測序的有序化，基因突變的單分子診斷和蛋白質(zhì)的納米芯片等。2.蛋白質(zhì)的STM研究功能蛋白細胞色素C2。蛋白質(zhì)的STM研究（紅素氧還蛋白分子）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 的基本原理與STM類似，在AFM中，使用對微弱力非常敏感的彈性懸臂上的針尖對樣品表面作光柵式掃描。當針尖和樣品表面的距離非常接近時，針尖尖端的原子與樣品表面的原子之間存在極微弱的作用

44、力（10-1210-6N），此時，微懸臂就會發(fā)生微小的彈性形變。針尖與樣品之間的力F與微懸臂的形變 之間遵循虎克定律：F=-k*x ，其中，k為微懸臂的力常數(shù)。所以，只要測出微懸臂形變量的大小，就可以獲得針尖與樣品之間作用力的大小。 “恒力”模式 針尖與樣品之間的作用力與距離有強烈的依賴關系，所以在掃描過程中利用反饋回路保持針尖與樣品之間的作用力恒定，即保持為懸臂的形變量不變，針尖就會隨樣品表面的起伏上下移動，記錄針尖上下運動的軌跡即可得到樣品表面形貌的信息。這種工作模式被稱為“恒力”模式（Constant Force Mode），是使用最廣泛的掃描方式。 “恒高”模式 AFM的圖像也可以使用

45、“恒高”模式（Constant Height Mode）來獲得，也就是在X，Y掃描過程中，不使用反饋回路，保持針尖與樣品之間的距離恒定，通過測量微懸臂Z方向的形變量來成像。這種方式不使用反饋回路，可以采用更高的掃描速度，通常在觀察原子、分子像時用得比較多，而對于表面起伏比較大的樣品不適用。 AFM的優(yōu)點： 原子級水平 各種條件下 樣品制備簡單 真的表面結(jié)構(gòu) 實時動態(tài)快速過程 小尺寸樣品的加工實驗條件 基底選擇 探針 制樣AFM在結(jié)構(gòu)生物學中的應用 生物大分子（130nm的DNA的片段） 細胞、細胞膜、人工質(zhì)子體 病毒、細菌、微生物?？乖⒖贵w 生物表面事件的實時原位監(jiān)測（動態(tài)）10-100S（細胞的分裂和RNA翻譯）: 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