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  • 516 MICROSCOPY, SCANNING TUNNELING

    Encyclopedia of Medical Devices and Instrumentation, Second EditionCopyright # 2006 John Wiley & Sons, Inc.

    MICROSCOPY, SCANNING TUNNELING

    , edited by John G. Webster

    VIRGINIA M. AYRESLALITA UDPAMichigan State UniversityEast Lansing, Michigan

    INTRODUCTION

    Four years after its invention in 1982 (1), the scanningtunneling microscope (STM) was awarded the 1986 NobelPrize for physics, one of only four such prestigious awardsgiven for a truly significant contribution to scientificinstrumentation. Since then, the family of scanning probemicroscopy (SPM) techniques, which includes scanningtunneling microscopy, atomic force microscopy (24), mag-netic force microscopy (5), near-field optical microscopy (6),scanning thermal microscopy (7), and others, has revolu-tionized studies of semiconductors, polymers, and biologi-cal systems. The key capability of SPM is that, through acontrolled combination of feedback loops and detectorswith the raster motion of piezoelectric actuator, it enablesdirect investigations of atomic-to-nanometer scale phe-nomena.

    Scanning probe microscopy is based on a piezoelectric-actuated relative motion of a tip versus sample surface,while both are held in a near-field relationship with eachother. In standard SPM imaging, some type of tip-sampleinteraction (e.g., tunneling current, Coulombic forces, mag-netic field strength) is held constant in z through the use offeedback loops, while the tip relative to the sample under-goes an xy raster motion, thereby creating a surface mapof the interaction. The scan rate of the xy raster motionper line is on the order of seconds while the tip-sampleinteraction is on the order of nanoseconds or less. The SPMis inherently cable of producing surface maps with atomicscale resolution, although convolution of tip and sampleartifacts must be considered.

    Scanning tunneling microscopy is based on a tunnelingcurrent from filled to empty electronic states. The selectiv-ity induced by conservation of energy and momentumrequirements results in a self-selective interaction thatgives STM the highest resolution of all scanning probetechniques. Even with artifacts, STM routinely producesatomic scale (angstrom) resolution.

    With such resolution possible, it would be highly desir-able to apply STM to investigations of molecular biologyand medicine. Key issues in biology and medicine revolvearound regulatory signaling cascades that are triggeredthrough the interaction of specific macromolecules withspecific surface sites. These are well within the inherentresolution range of STM.

    The difficulty when considering the application of STMto molecular biology is that biological samples are non-conductive. It may be more accurate to describe biologicalsamples as having both local and varying conductivities.These two issues will addressed in this article, andexamples of conditions for the successful use of STM forbiomedical imaging will be discussed. We begin with anoverview of successful applications of STM in biology andmedicine.

  • MICROSCOPY, SCANNING TUNNELING 517

    Figure 2. STM image of portion of Hetero A-T double helix ofshowing base pairs. (Reproduced from Ref. 9, used withpermission.)

    SCANNING TUNNELING MICROSCOPY IN BIOLOGY ANDMEDICINE: DNA AND RNA

    The STM imaging for direct analysis of base pair arrange-ments in DNA was historically the first biological applica-tion of the new technique. An amusing piece of scientifichistory is that the first (and widely publicized) images (812) of (deoxyribonucleic acid) DNA were subsequentlyshown to correspond to electronic sites on the underlyinggraphite substrate! However, more careful investigationshave resulted in an authentic body of work in which thebase pairings and conformations of DNA and RNA aredirectly investigated by STM. One goal of these investiga-tions is to replace bulk sequencing techniques and crystaldiffraction techniques, which both require large amounts ofmaterial, with the direct sequencing of single molecules ofDNA and RNA. Two examples of DNA and RNA investiga-tion by STM are presented here. One is an investigation ofDNA and RNA structures, and the other is an investigationof DNA biomedical function.

    Recently reported research from the group at The Insti-tute for Scientific and Industrial Research at Osaka Uni-versity in Japan (13) has shown detailed STM images ofwell-defined guanine-cytosine (G-C) and adenine-thymine(A-T) base pairings in double- and single-stranded DNA.Four simple samples involving only G-C and only A-T basepairs in mixed (hetero) and single sided (homo) combina-tions were chosen for analysis (Fig. 1). These were depos-ited on a single-crystal copper (111)-orientation [Cu(111)]substrate using a technique developed specially by thisgroup to produce flat, extended strands for imaging. AnSTM image showing the individual A-T base pairs in thehetero A-T sample is shown in Fig. 2. Images of the overallstructures indicated repeat distances consistent with inter-pretation as the double helix. Images from mixed samplesof hetero G-C and hetero A-T are shown in Fig. 3. Thelarger structure is interpreted as hetero G-C and thesmaller as hetero A-T, which is consistent with X-raydiffraction data that indicates the A-T combination is morecompact.

    Only the double helix structure was observed for thehetero G-C samples. However, the homo G-C structures,

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    Figure 1. (a) Homo A-T, (b) Hetero A-T, (c) Homo G-C, and (d)Hetero G-C. (Figure adapted from Ref. 9, used with permission.)

    hetero A-T structures, and homo A-T structures wereobserved in two types, and the spot spacings and sizes ofthe second type would be consistent with interpretation assingle-stranded DNA. The observed presence or lack ofsingle-stranded configurations among the samples is con-sistent with the fact that hetero G-C has a higher melting(unraveling) temperature than the homo G-C and thus ismore difficult to unwind. Both hetero and homo A-T pairshave lower melting temperatures than either of the G-Cpairs. Images of both hetero A-T and Homo A-T samplesoften showed sizing and spacings consistent with inter-pretation as single-stranded DNA, in addition to observeddouble helix specimens. Thus, the presence/lack of single-stranded versus double helix images is consistent withknown melting temperature data for the C-G and A-T basepairings.

    The same group has also reported successful STMinvestigations of transfer-ribonuclic acid (t-RNA) (14). In

    Figure 3. Hetero G-C and Hetero A-T mixed sample. The largerspecimens are identified asHetero G-C, and the smaller specimensare identified as Hetero A-T. Both are in a double helixconfiguration. (Reproduced from Ref. 9, used with permission.)

  • 518 MICROSCOPY, SCANNING TUNNELING

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    Figure 4. (a) Model of t-RNA L-shapedconformation. (b) Model base pair arrangementin L-shaped conformation. (c) STM image of L-shaped conformation at physiological pH.(Reproduced fromRef. 10, usedwith permission.)

    Figure 5. (a) STM image of retinoic acid on a graphite substratecompared with its molecular model showing the aliphatic ringhead and polymeric tail. (b) STM image of retinoic acid binding tot-RNA with molecular model overlay. (Reproduced from Ref. 13,used with permission.)

    RNA, the base pairing is adenine-uracil (A-U) instead ofadenine-thymine (A-T). Also the backbone sugars areribose rather than deoxyribose, but are still linked byphosphate groups. The RNA is very difficult to synthesizeas a single crystal and consequently there is a very limitedamount of X-ray diffraction data available for RNA. Littleis known about its variations, and therefore direct inves-tigations of single molecule RNA would add much to ourknowledge.

    Transfer RNA is a small RNA chain of 7493 nucleo-tides that transfers a specific amino acid to a growingpolypeptide chain at the ribosomal site of protein synthesisduring translation (15). It has sites for amino acid attach-ment, and an anticodon region for codon recognition thatbinds to a specific sequence on the messenger RNA (m-RNA) chain. It has a partial double-helix structure eventhough it has only one chain, because the single RNA chainfolds back, and loops back, on itself, as shown in Fig. 4a.

    X-ray diffraction studies (16) have indicated that thet-RNA structure may often assume an L-shaped conforma-tionwith a long and a short arm. Amodel of the EscherichiaColi lysine t-RNA macromolecule used by the group for itsSTM studies is shown in Fig. 4a and b. It shows both theL conformation and the underlying loop and base pairchemistry.

    Using STM, the group was able to directly image the Lconformation as shown in Fig. 4c. In addition to the firstdirect statistical data on the lengths of the long and shortarms, obtained from analysis of several STM images, ananalysis of the influence of pH on conformation was alsocarried out. Current investigations are focusing on biofunc-tion research issues in addition to structural researchissues, using STM to directly image the coupling of theim