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Scanning Tunneling Microscopy of Biological Molecules on Pt(ll1): From 100 to 5 X lo6 Da R. J. Wilson,' K. E. Johnson, D. D. Chambliss, and B. Melior IBM Almaden Research Center, San Jose, California 95120-6099 Received June 21,1993. In Final Form: August 30,1993@
We have performed ultrahigh vacuum (UHV) scanning tunneling microscopy studies of biological molecules by developing a UHV compatible cell wherein a drop of ultrapure water containing nanograms of molecules is evaporated from a strongly chemisorbing Pt(ll1) surface in an Ar ambient. Images are presented for hydroquinone; oligomers including pentaphenyl ether, enkephalin, and sodium dodecyl sulfate;large homopolymers of lysine and glycine; the proteins calmodulin,tropomyosin, and immunoglobin G; and double- and single-stranded plasmid DNA. In all cases the apparent height is only a few A. Imagingpropertiesunderstandably vary from clearly resolved internalmolecular structure for hydroquinone to diffuse, artifact-prone images for molecules which exceed tens of angstroms in thickness. These data do not support claims for atomic resolution imaging of complex biomolecules.
Introduction The ability of scanning tunneling microscopy (STM) to image the internal structure of molecules has aroused considerableinterest for the last few years. Several studies using graphite surfaces have shown that many small molecules form packed arrays which can be imaged at very high resolution.14 Experiments on small aromatic molecules on metal crystal surfaces, prepared and measured under ultrahigh vacuum (UHV) conditions, also revealed an internal structure which, in some cases, did not require molecular packing to eliminate motional instabilities.@ A number of papers have also appeared which asserted that high-resolution images could be obtained even for larger molecules such as DNA and proteins, leadingto claimsfor sequencing and for structural observations of biomolecules. Unfortunately, little progress has been made and poor adhesion of biomolecules to inert supports such as graphite has been blamed. As a result of this poor adhesion, the molecular coverage in early work was not easily controlled so that workers were evidently forced to select rare images, which they believed to represent molecules, from an unknown background of possible impurities and surface d e f e ~ t s Reproducibility .~ has been obtained more recently in atomic force microscopy (AFM) and STM studies of plasmid DNA which is chemically linked to mica or gold surfaces by ionic interactions which may involvesmall binder mo1ecules.g-10 For large molecules the price of this reproducibility apparently involves a degradation of lateral resolution which obscures atomic scale features. On the other hand, e Abstractpubliihedin Advance ACSAbstracts, October 15,1993.
(1) Foster, J.; Frommer, J. Nature 1988,333, 542. (2) Rabe, J. P. Phys. Reu. Lett. 1991, 66, 2096. (3) Smith, D.P. E.; Horber, J. K. H.; Binnig, G.; Nejoh, H. Nature 1990,344,641. (4) Ohtani, H.; Wileon, R. J.; Chiang, S.;Mate, C. M.Phys. Res. Lett. 1988.60.2398. (5) Lippel, P. H.; Wilson, R. J.; Miller, M. D.;Wall, Ch.; Chiang, S. Phys. Reu. Lett. 1989, 62, 171. (6)Hallmark, V. M.; Chiang, S. Phys. Rev. Lett. 1991,66, 48. (7) Clemmer, C. R.; Beebe, T. P., Jr. Science 1991,251,640. (8) Hansma, H. G.; Vesenka, J.; Siegrist, C.; Kelderman, G.; Morrett, H.; Sinsheimer, R. L.; Eliigs, V.; Bustamente, C.; Hansma, P. K. Science 1992,256, 1180. (9) Bustamante, C.; Vesenka, J.; Tang, C. L.; Rees, W.; Guthold, M.; Keller, R. Biochemistry 1992,32, 22. (10)Alliaon, D. P.; Bottomley, L. A.; Thundat, T.; Brown, G. M.; Woychik, R. P.; Schick, J. J.; Jacobson, K. B.; Warmack, R. J. Proc. Natl. Acad. Sci. 1992,89,10129. Thundat, T.;Allison, D.P.; Warmack, R. J.; Brown, G. M.;Jacobson, K. B.; Schick, J. J.; Ferrel, T. L. Scanning Microsc. 1992, 6, 911.
a number of recent reports have demonstrated reproducible high resolution imaging of packed arrays of individual DNA bases on graphite.11J2 The factors which limit resolution remain poorly understood, and considerable confusion still shrouds the origin of the image contrast, or effective height, of these nominally insulating molecules. Clearly a better understanding of this contrast, and of conductivity anomalies proposed to allow one to image thick molecular layers, is important to continuing STM studies of biomolecules. One cannot deny that a simple extension of atomic resolution STM to complex polymers might have significant impact. Obviously a tangled heteropolymer, whose flexible side chains differ in surface binding and are sterically constrsined by neighboring residues, is more complex than a simple molecule which crystallizes or strongly binds in a specific adsorption site.13 Still, these molecules may not image so differently from small molecules if they can be spread into nearly 2-D structures on surfaces. Since large molecules would not be expected to universally form packed arrays, it appears advantageous to investigate surfaces which can immobilize each subunit. Observations of isolated small molecules a t room temperature (RT) have been reported for aromatics on Cu and Pt surface^^*^ but not for inert molecules, such as hydrocarbons, or inert supports like Au(111)140rgraphite. Unfortunately, the diversity of functional groups in biological molecules makes strong bonding of each subunit unlikely so that high resolution is not guaranteed even for ideal surface conformations. For tangled molecules, which possess substantial three-dimensional character, the situation is even less clear. Multilayer films of copper phthalocyanine,5 for example, failed to yield high resolution data at coverages above 1 monolayer (ML). At coverages of several ML even the step structure disappeared and images became featureless, suggesting continuous contact between the tip and the insulating film. On this basis one might expect to be able to observe untangled polymer chains, with lateral resolution being dependent on the binding of the monomers as influenced (11) Allen, M. J.; Balooch, M.; Subbiah, S.;Tench, R. J.; Siekhaus, W.; Balhorn, R. Scanning Microsc. 1991,5,625. (12) Heckl, W. M.; Smith, D. P. E.; Binnig, G.; Klagges, H.; Hansch, T. W.; Maddocks, J. Proc. Natl. Acad. Sci. 1991,88,8003. (13) Wattenbarger, M. R.; Chan, H. S.; Evans, D.F.; Bloomfield, V. A.; Dill, K. A. J. Chem. Phys. 1990,93,8343. (14) Wilson,R.J.;Meijer,G.;Bethune,D.S.;Johnson,R.D.;Chamblise, D.D.;deVries, M. S.; Hunziker, H. E.; Wendt, H. R. Nature 1990,348, 641.
0743-7463/93/2409-3478$04.00/0 0 1993 American Chemical Society
STM of Biological Molecules by constraints imposed by the overall atomic structure of the molecule and substrate. In tangled or overlapping regions, the resolution would diminish but the observed heights should provide some insight into proposed tunneling condution anomalies. The present work is aimed at obtaining some insight into the magnitude of these problems and at determining what useful work might be possible in the context of experimental realities. To this end, we present images of one small molecule and extend our observationsto several oligomers and larger molecules of proteins and DNA. Most of these molecules are briefly described in introductory biochemistry texts.16
Experimental Apparatus To deposit the molecules used in this study, which range in molecular weight from 100 to lo7, a simple ultrahigh vacuum (UHV) compatible solution cell (SC)was constructed where a drop (2 rL) of water bearing dilute sample molecules (1pg/mL) can be deposited on and evaporated from a clean single crystal. This chamber is attached to an instrument16 which is equipped with a variety of sample transfer, surface preparation, and analysis facilities. Storage solutions of sample molecules are made by dissolving Sigma chemicals at 0.1 mg/mL concentrations in pyrolytic water.17 Storage solutions were held cold in glass ampules sealed by Teflon compression fittings and backfilled with Ar. A dilution to microgram per milliliter levels was made by metering a small volume of the storage solution into an identical container of pyrolytic water. This working solution was then transferred through a coaxial Teflon line, wherein the return flow jackets the feed line to reduce impurities, into a glass and Teflon valve on the SC where it was purged with LN trapped He for 30 min to remove dissolved gases. Next a crystal, cleaned by standard UHV techniques including cycles of Ar sputtering, 0 2 exposure,and annealingat elevated temperatures, was transferred into the SC under UHV conditions. Valves were used to isolate the cell from the UHV system, and the cell was backfilled with 30 Torr of Ti-purified Ar to limit the evaporation rate of the droplet to be applied. The solution valve was then opened to allow a few microliters into a thin Teflon tube which conducted the liquid to the sample face. The resulting droplet wet a 0.5 cm2 area and evaporated from the surface in about 1min. The cell was then evacuated and the sample was returned to the UHV instrument for characterization and STM measurements. Scanning Auger microscopy (SAM)has been a critical monitor of the cleanliness of solvent only depositions and of the concentrations necessary to produce the desired coverage. For Au(ll1) surfaces, SAM showed that dissolved adsorbates were primarily concentrated in the region last occupied by the evaporating drop. Higher concentrations were used so that C could be detected on the surface by SAM, but repeatable images of molecules were not obtained with STM. Cu(ll1) surfaces exposed to clean water showed strong oxygen contamination18 and appeared rough and disordered in the STM. Considerable refinements of the original apparatus were required to solve contamination problems for the reactive Pt(ll1) surface. These problems included impurities in the water, backfill and purge gases, and contaminants which were displaced by water vapor from the UHV chamber walls onto the Pt(ll1) sample.19 Ultimately, no contamination was detected by SAM and adsorbates were found to bind reasonably uniformly over the exposed area. A fuller account of the instrumentation and methods will be published elsewhere. Many STM measurements of different (15) Stryer, L. Biochemistry; W. H. Freeman: New York, 1988. (16) Chiang, S.; Wilson, R. J.; Gerber, Ch.; Hallmark, V. M . J. Vuc. Sci. Technol., A 1988,6, 386. (17) Pyrolytic water waa generously provided by P. Ross. This water, which is distilled over Pt gauze at 900 O C in an 0 ambient to remove
impurities, could be ueed for several weeks provided it waa stored in an Ar-purged Blase bottle. (18)Stickney, J. L.;Ehlers, C. B.; Gregory, B. W. Langmuir 1988,4, 1368. (19) We are indebted to A. T. Hubbard for pivotal diecusaions of
methods for obtaining clean, water-exposed Pt surfaces.
Langmuir, Vol. 9, No. 12,1993 3479 molecules on this Pt(ll1) crystal, alreadyused in this instrument for some years! have convinced us that the imaged structures result from the applied chemicals. These extended efforts and advanced instrumentation have required us to confidentlypresent representative data, rather than selecting advantageous but weproducible results which we regard aa artifacts, even though the data might otherwise seem unconvincing. Finally, we note that it would be preferable to control electrochemical potentials, ionic strength, and pH, but we have chosento minimize the effects of impurities by omitting salts and buffers where possible.
Brief Review Before proceeding to the data it is helpful to review some aspects of adsorption of organic molecules from solution. The evaporated droplet deposition method is expected to lead to substantial variations in the physical processes which occur during adsorption because of the wide variability in solution diffusion of the molecules which we address. Since diffusion constants for spherical particles in aqueous solutions are typically 5 X 10-2/ d(mm2/s),where d is the diameter in A, small molecules diffuse across the 0.1 mm thick droplet before evaporation and primarily bind from bulk liquid. Large plasmid DNA molecules will diffuse negligibly during this time so that large molecules will be concentrated near the surface of the evaporating solvent when they arrive at the crystal face. Charged groups are often essential for the aqueous solvation of large molecules. Molecules which are charged in solution can be neutralized by reattachment of counterions during the final stages of evaporation or they may be neutralized, along with a remote counterion, when both attach to the Pt surface. Upon evaporation, it is highly probable that neutralized, surface bound counterions will accumulatenear molecules as a result of the partially ionic character of nominally neutralized species. Surface bound salts may appear as isolated debris, condense to form ordered or disordered arrays, or rapidly diffuse on the surface. It is well known that strong forces between molecules can give rise to solution aggregation effectswhich can result in the formation of surface films and of bulk structures like micelles or precipitates. Adsorbed molecules may similarly interact with one another and the diminishing water layer may exert strong forces which are associated with the desire of charged groups to remain solvated and of hydrophobicgroups to exclude solvent. In view of this complexity, it is fortunate that substantial work on Pt(ll1) has resulted in reliable structural models for specific chemisorptionof many small molecules. The effects of bonding on the vibrational spectra of specific functional groups within molecules have been deduced from electron energy loss spectroscopy (EELS) studies of thin electrochemically deposited film^.^ It is generally believed that, at room temperature, the Pt(ll1) surface does not break C-C or C-N bonds, although such breakage is possible at low coordination Pt atoms at surface steps.m*21 Strong attachments of organic molecules to atomically flat terraces are obtained for C=C double bonds in chains or cyclic configurations, but small saturated hydrocarbons are not strongly,or irreversibly, adsorbed on this surface.20 H atoms are often displaced from alcohols, thiols, low coordination N atoms, and sometimes from carboxylic acids to result in direct coordination of Pt to the molecule.20 Undissociated nonaromatic molecules can also adsorb, as evidenced by structural models for several amino acids on (20)Hubbard, A. T. Chem. Rev. 1988,88,633. (21)Somorjai,G. A.Chemistry in WoDimemions: Surfaces;Cornell University Press: Ithaca, N.Y.,1981.
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Figure 1. STM images of solution-deposited hydroquinone: (a) A wide scan image reveals an unidentifiable structure for a solution concentration of 2 pg/ml. (b) At higher magnification,there is evidence of chain structure and individual molecules can be identified. (c) When deposited from less concentrated solutions, condensed chainlike structures are still formed. (d) At still higher magnification internal molecular structure becomes apparent. The tunnel current was fixed at about 0.5 nA throughout this work and the tip bias held at Vt = 0.02-0.1 V with no notable polarity dependence. Bar is 50 A.
Pt(lll).n Even with these models, we have little insight intothe motion of molecules on surfaces, either in vacuum or under solution. Rapid surface diffusion can lead to the formation of aggregates which may or may not be stable. At present, it is not possible to predict whether such diffusion is impossible for small, covalently attached molecules or for large, weakly bound molecules. Even in the absence of diffusion,the positions of parts of molecules may fluctuate at room temperature if the stabilizing forces are as weak as hydrogen bonding or van der Wad’s interactions. These concerns apply strongly to portions of molecules which are not in contact with the surface and which may deform by weakly hindered rotations acting over several bonds.
As a f i t example of system performance, we show images for solution-deposited hydroquinone molecules. Figure la showsa large scale image which is representative of the image sizes used later for macromolecules. The present image appears as a surprisingly complex network
which does not suggest any underlying order. A monoatomic 2.26 high Pt(ll1) step, which is present because of the deviation of the mechanically polished crystal face from the ideal (111)orientation, sets the vertical gray scale. At higher magnification, Figure lb, it can be seen that this deposit is comprised of peculiar chains which occasionally pack densely, but which also enclose large vacant regions. An image at a lower coverage, Figure IC,still shows chains and large vacant regions, so that it appears likely that hydroquinone molecules, bound by both covalent linkages and strong aromatic interactions, can still diffuse and aggregate on the solution-covered Pt(111)surface at room temperature. Comparisons with vapor phase deposits, or of solution deposited quinone, could illuminate the interesting role of solution effects. At even higher magnifications, Figure Id, several different molecular orientations can be easily distinguished. Apparent chain formation clearly involves staggered end to end aggregation. The molecular images show a strong resemblance to the ?r lobes expected for quinone, and it is believed that the phenolic hydrogens are rem0ved.~3The staggered end to end aggregation, on the other hand, suggests that chain
(B)Stem,D. A; Salaita, G. N.; Lu, F.; McCargar, J. W.; Batina, N.; Frank, D. G.; Laguren-Davidson, L.; Lin, C.; Walton, N.; Gui, J. Y.; Hubbard, A. T. Langmuir 1988,4,711.
(23)Lu, F.;Salaite, G. N.; Laguren-Davidson,L.;Stem,D. A;Wellner, E.; Frank,D. G.; Batina, N.; Zapien, D. C.; Walton, N.; Hubbard, A. T. hngmuir 1988,4, 637.
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formation may result from hydrogen bonding of the phenolic H in solution prior to dissociation. If these H atoms are removed, these structures could still arise if coordination of an 0 atom removes some charge from a target Pt atom which then interacts indirectly with the 0 dipole of an adjacent molecule. The high density ordered domains are in reasonable agreement with earlier low energy electron diffraction (LEED)observations of a 3 X 3 overlayera with the molecules apparently slightlyrotated relative to the unit cell in such a way that two domains are formed. The tall defects observed in many of these images may well arise from edge bound molecules which are known to exist at high coverages. We next proceed to a study of a few oligomers. The first case is a pentameric phenyl ether with an undisclosed distribution of meta and para linkages (Santovac from Monsanto) which was deposited in UHV by using the room temperature vapor flux from a small liquid film. This molecule is somewhat similar to a polymeric HQ and one would hope to image strings of flat lying rings. A wide scan image, Figure 2a, indicates that many conformations are present. Many molecules appear tangled and somewhat taller. These molecules are all quite immobile on the surface so that much of the tangling may be frozen in by strong binding. However, an inspection of the extended molecules in this image reveals that they are nearly all extended along directions related by 3-fold rotations, suggestingthat quasi-flat-lyingconformationsare possible only for some orientations of the molecules relative to the %fold Pt(ll1)surface. Figure 2b is a higher magnification image which includes one feature, marked with an arrow, which is indeed suggestive of five linked benzene rings. However, other molecules which appear extended often still appear diffuse. To understand the diffuse structure of extended molecules, one must realize that adsorption generally involves a specific registration of the molecule relative to surface Pt atoms. If we choose a given ring in a flat-lying molecule to occupy the optimal site, then the positions of neighboring rings are constrained by the length, orientation, and meta-para nature of the ether linkages. If these positions do no coincide with preferred binding sites, then the flat lying conformation may distort since the rings can rotate about weakly hindered O-ring bonds. The %fold rotational alignment of extended molecules may thus arise from “molecular epitaxy” which will vary for different bond rotation angles or distinct combinations of meta and para linkages. This “epitaxy” is usually imperfect so that surface-induced distortions may prohibit conformations where all rings lie flat. In this case the energetics and activation barriers of more complex conformations must be considered to establish the effects of thermal excitations or tip-related forces on librational motion. It should be realized that the surface is not creatinginternal molecularmotion, which is certainly active in solution, but rather that it may be unable to suppress it when numerous nearly degenerate modes are involved. At still higher magnifications, Figure 2c, we see that some fine structure can be detected within virtually every molecule, so that it might be possible to assign specific conformations to individual molecules and to better understand possible motional effects on diffuse contrast. Without further systematic studies for specific meta-para combinations, the variability of surface conformations obscures the variability associated with the mixed meta and para linkages. Evidently, atomic resolution imaging of single conformations of isolated monomers need not imply simple conformations or images for even short flexible polymers.
1
I 8
Figure2. (a)An image of a field of vapor-depositedpentaphenyl ether molecules reveals a cornucopia of conformation. (b) At higher magnification, one finds varying degrees of internal structurein differentmolecules. (c)At still higher magnification, structure is detected within most molecules. Vt = 0.1-0.5 V, bar = 50 A.
As a second case, we consider leucine-enkephalin (tyrosine-glycine-glycine-phenylalanine-leucine), which is a bioactive peptide whose conformation is of interest.24 -
(24) Nayeem,A,; V i a , J.; Scheraga, H.A. J. Comput. Chem. 1991,12, 594.
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The strong bonding of Pt(ll1)to the aromatic side chains of tyrosine and phenylalaninemshould help to immobilize these molecules. The image in Figure 3a shows a region where one observes small but variably sized clumps which cannot be readily separated into single molecules. In the adjacent frame, Figure 3b, one finds a much larger patch where molecules appear to have condensed into an island structure. Figure 3c shows a magnified view of this island which does reveal a reasonably sharp structure, but one cannot distinguish the individual molecules because of strong clustering and inequivalence. We have been quite puzzled as to the origin of this type of disordered cluster formation. It is known that leucine-enkephalin can be crystallizedin certain solvents in a complex type of planar @-sheetstructure25 whose unit cell involves multiple inequivalent molecules with the aromatic sidegroups extending into the wide region which separates the weakly coupled sheets. However, it is not clear that forces associated with crystallization can operate for molecules which are strongly bound to the Pt(ll1)surface. We have noted similar clustering phenomena for several other molecules and have noted that all these solutions showed a noticeable tendency to form stable foams during various purging operations, especially at 1mg/mL concentrations. Such foams are associated with the presence of surface films involving the formation of lamellae wherein an aqueous layer is confined by amphiphilic molecular films on both surfacesof bubbles within the foam.% Foam films can then be stabilized by the repulsion between ionized hydrophilic groups which are directed into the film while solvent exclusion of the hydrophobic moieties maintains the surfaceexcessconcentrations. At concentrations below that required for saturating the surface films, these foams are unstable, but one must still consider the possibility that the surfacefilms aggregate laterally to form structures which have been termed “hemi-micelles” which can essentially plate onto the Pt(111)surface. Alternatively, it is possible that these aggregates are associated with solvent-exclusion-drivenclustering of diffusing surface bound species, essentially “hemimicelle” formation at the metal-water interface. This will be further influenced by evaporation, when hydrophilic groups of surface bound, partially solvated molecules densify to share diminishing solvent. Similar phenomena must operate for many surfactant molecules and we therefore examined the detergent sodium dodecylsulfate (SDS)as deposited from 1pg/mL solutions. This amphiphilic molecule aggregates in solution when present above the critical micelle concentration (8 mM), but for these dilute (4 p M ) samples of rapidly diffusing molecules, surface films will accommodate many of the solute molecules prior to bulk micelle formation.% STM images, Figure 3d, show that SDS deposition does lead to both small and rather large islands. There is some internal structure in these islands but we have not obtained any high resolution images which reveal structure at the molecular level. It is noteworthy that different solution handling techniques, such as contacting the Pt(ll1) surface to an equilibrated bulk liquid surface, would eliminate some of the ambiguities associated with manipulating solutions whose content can be largely accommodated in surface films. In an optimistic view, this method would provide significant new insight into lateral aggregation of unsaturated surfactant films. These images reveal a tremendous diversity in imaging characteristics and aggregation phenomena for different ~
(25)Karle, I. L.; Karle, J.; Mastropaolo, D.; Cameraman, A.; and Cameraman, N. Acta Crystallogr. 1983, B39,625. (26) Roaen, M. J. Surfactants and Interfacial Phenomena;John Wiley and Sons: New York, 1978.
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1
Figure 3. (a) Images of molecules of solution-depositedacetate salt of leucine-enkephalin. (b) In some regions, more dense packing suggests some intermolecular interactions. (c) Within this island high resolution was obtained, but the images are too complex for interpretation. Vt = 0.4 V for (a-c). (d) Image showingan irregular dense island observedfor solution-deposited sodium dodecyl sulfate. Vt = 0.2 V, bar = 100 A.
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molecules which anticipate some of the difficulties to be expected for complex heteropolymers. One common theme in measurements for all of these oligomers is that the attainment and interpretation of high resolution images for flexible polymer molecules are obstructed by the appearance of many conformations which often show only diffuse structure and by aggregation phenomena which result in the formation of dense, disordered clusters.
Synthetic Biopolymers In order to extend these measurements to larger molecules without compromising the purity of the solutions, which is uncertain for several of the biomolecules to be discussed later, we examined several high-purity organicallysynthesized a-L-homopolypeptideswith numbers of monomers ranging from 100 to 550.27 Images obtained for deposition of 550-men of poly(L-lysine-HBr), whose amine-terminatedside chain should bind are shown in Figure 4a,b. In dilute neutral solution these moleculesare expected to have a random coil configuration with the chain somewhatextended over persistence lengths determined by repulsiveinteractions between charged side chains. Yet these molecules appear here in variably sized compact clusters which we believe to represent single molecules. To confirm this view, we have used solutions containing 0.05 pg/mL to obtain these images, where such features were found to be separated by thousands of angstroms, so that condensation of multiple molecules should be highly unlikely. In addition, since a single molecule in the extended &conformation would have a maximum length of about 2000 A, 500 A diameter hemicirclesare reasonably consistent with 2-D folded chain structures involving a 20 A wide single molecule. A distinctive feature of these data is that all clusters appear to nestle against a step edge and occasionally spill over onto an adjacent terrace, implying substantial mobility to allow accommodation to the substrate step and terrace morphology. Such intramolecular condensation can be attributed to the absence of strong chemisorption and the presence of significant surfactant character, where the backbone and four saturated carbons of the side chain form a hydrophobic counterpart to the ionized terminal side-groupamines. Figure 4c showsa higher magnification view of one feature shown in Figure 4a, which eliminates pixel limitations which can result in misleading images. Stable internal structure on a 20-1(lateral scale was repeatably obtained and is consistent with the observed molecular area. No molecular displacements were observed from image to image but there are a few clues which might help us rule out possible multiple imaging effects. In an attempt to display the chainlike character of these molecules, images were obtained for 120-mers of a 4:l random copolymer of L-lysine-HBr and L-tyrosine, which were included for solubility and strong chemisorption, respectively. Figure 4d shows data for a much higher coveragewhich is near the upper limit for recording useful data. These molecules now appear as narrow partially overlappingchains whose contour length is consistent with that of extended 120-mers (400 A) and many molecules appear to extend over surface steps. Evidently, the addition of strongly binding side groups does eliminate rearrangements which allow adjustment to surface topography* It is important to question the possible role of salts and dissociation in these homopolymer images. We have (2'7) Faaman, G. D. PoZy a-fiAmino Acids, MarcelDekket: New York,
1967.
Figure 4. (a, b) Wide scan images show two features observed following aqueous deposition of poly-(L-lysine-HBr). (c) Image showing a higher magnification image of (a). (d) Images of a random copolymer containing one L-tyrosine for three L-lysineHBr obtained for 2 pg/mL. Bias voltages of 0.5-1 V seemed to reduce contact problems for polymers. Bar = 300 A.
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solution. It is well known that polyglycine is not readily water soluble and that aggregates of antiparallel @-sheet or polyglycine I1structures can be formed by casting films from organic solvents or by precipitation from high salt aqueoussolutionsby dilution, respectively.m-B We believe our “solution” actually involves both surface films and suspended aggregateswhose size distribution is accessible to STM. These large aggregates did show quasi-periodic corrugations at molecular scales, Figure 5c, but it was not possible to identify individual moleculesin the two allotted runs. These aggregates again appear to adapt to step shapes, so that some mobility appears to be involved. The similarity of these images to those of the lysine-based polymers argues against salt artifacts. The primary conclusion of this homopolymer work is that the presence of multiple conformations and diffuse character of images of short oligomers appears to apply to the both the chain structure and the side groups. The resulting imagesare socomplexthat there is no clear reason to hope for any applications of atomic-scale resolution. Nominally straight sections of molecules appear as chains with a width comparable to twice the side group lengths, but complex conformations frequently obscure even this level of interpretation. This resolution is not suitable for many purposes, but it can be competitive with other microscopies.
Proteins For a random sequence polypeptide about one in four amino acids will have strongly binding aromatic or hydroxyl side groups, much as for the mixed copolymer previously discussed. To test whether these results can be extended to real proteins, we have examined calmoddin (CaM),a singlechain 140-merwhich lacks disulfidebridges. CaM strongly binds Ca and serves as a Ca sensor when associated in many coenzyme complexes.m The structure of the crystalline Ca bound protein (CaM-Ca) is k n 0 ~ n 3 l and the solution conformation has been studied by NMR.32 There is much interest in the structure of CaM complexes formed with other peptides33and dyes.34 Figure 6a shows a wide scan image of many CaM molecules on the Pt(111) surface. Considerable debris is evident and the supplier warns that Ca may be present. This submonolayeramount of Ca should not give the Ca-bound protein in our initial solutions, but it is possible that Ca is bound during evaporation. In spite of the debris, which is generally increased for biological materials relative to the organically synthesized polymers, it is not difficult to find similar looking, relatively untangled chains which show widths at the 20-Alevel,Figure 6b. In the crystal structures of CaMCa an a-helical chain is folded into a dumbbell shape which is only 70 A long. In these images there is no evidence for the CaM-Ca dumbbell and the molecules appear quite extended with apparent heights of only a few angstroms, which are not in accord with the true heights of a-helical structure. In an effort to preserve solutionstructure during Figure 5. (a, b) Wide scan images showing a range of islands observed for polyglycine. (c) Fine structure observed for one of the aggregates in (a). Vt= 1.0 V, bar = 500 A.
therefore examined images obtained by depositing 100mers of polyglycine. We found a broad size distribution of deposits, which cannot be associated with individual molecules, as shown in Figure 5a,b. Although foaming properties were not observed, and not expected for purely hydrophobic molecules, the cloudy nature of the storage solution,which was reduced by sonicatingfor a few minutes at 50 “C, implied the existence of aggregates within bulk
(28) Bamford,C. H.; Brown,L.; Cant, E.M.;Elliott,A.; Hanby, W. E.; Malcolm, B. R.Nature 1955,176,396. (29) Kuroki, S.; Ando, I.; Shoji, A.; Ozaki, T. J. Chem. Soc., Chem.
Commun. 1992,5,433. (30) Means, A. R.; Dedman, J. R. Nature 1980,285,73. (31) Babu, Y. S.; Sack, J. S.; Greenhough,T. J.; Bugg, C.E.;Meane, A. R.;Cook,W. J. Nature 1985,315,37. Babu, Y. S.; Bugg, C. E.;Cook, W.J. J . Mol. Biol. 1988,204, 191. (32) Ikura, M.; Spera, S.; Barbato, G.; Kay, L. E.;Krinks, M.;Bar, A. Biochemistry 1992,31, 5269. (33)Roth, S. M.; Schneider, D. M.;Strobel, L. A; Van Berkum, M.F. A.; Means, A. R.; Wand, A. J. Biochemistry 1992,31,1613. (34) Shiner,R. F.; Albaugh, S.; Nenortas, E.; N o d , L. BiopoZymem 1992, 32, 73.
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4
Figure 6. STM images of the protein calmodulin. (a) A wide scan image showing variably tangled and overlapped molecules. (b) A higher magnification image taken near the center of (a) showing a few similaruntangled structures. (c) Images taken for freeze-dried deposition. V, = 0.5-1.0 V, bar = 200 A.
adsorption on the surface, we have pumped on the evaporating droplet to obtain a freeze-dried deposit. The image of Figure 6c illustrates more clusteringof individual molecules, which in some cases suggests end to end aggregation. This clusteringmay result when slowfreezing sequesters the proteins at the boundaries of ice grains.
Langmuir, Vol. 9, No. 12, 1993 3485
Although one can identifyan X-shapedfeature with lateral dimensions appropriate to the dumbbell conformation, this feature is almost certainly an impurity. The bovine calmodulin used in these experiments is heterogeneous because of the substantial posttranslational modifications which importantly modify the functions of this enzyme. Recombinant CaM would eliminate this heterogeneity, but this realization suggests that it might be possible to coarsely mark and locate specific posttranscriptional modifications using distinctive markers like 1nm colloidal gold particles, which would appear distinctiveas the tallest features in these images. A simple test of our ability to discern any vestige of macromolecule architecture was conducted in studies of a simple extended dimeric protein. Figure 7ashow several molecules of chicken tropomyosin, which consists of two identical chains of about 330 amino acids. In solution each chain is largely a-helical and the two identical a-helices coil loosely about one another, with six or seven half-turns over the 420-A length of the tropomyosin molecule.36s36 This coiled coil structure is stabilized in solution by the packing of hydrophobic knobs into holes in the adjacent helix. The domainant motif in these images is perhaps best described as a distorted "8*.This geometry suggests that the two chains tend to separate on the Pt(111)surface in vacuum, where the hydrophobic stabilization is likely replaced by interactions which try to bond each side group to the surface. The picture of adsorption in this case is that binding to the surface separates and distorts the coiled a-helices. The separation of the chains is topologicallyobstructed by the initial intertwining and by possible interchain covalent attachments at the disulfide bridges. Given the small number of turns of this coiled coil, it is not difficult to rationalize the "8" motif. Internal structure is confused in this case because of tangling, but 20-A detail is still attainable in untangled regions, Figure 7b. However, one is again faced with the fact that the observed heights are only on the order of a few angstroms sothat essentiallyno insight can be obtained into the real 3-D structure of these molecules. These data may be compared with TEM images of shadowed trop o m y o ~ i nor~ ~with those of stained, quasi-crystallized tropomyosin structures3e3 to gain some insight into the severe limitations on both techniques. As a cautionary note we include two images, Figure 7c,d, which were unexpectedly obtained for a sample which was nominally nonacetylated a-tropomyosin, whereas the previous data were for a mixture of posttranscriptionally modified a! and 6 homodimers. This sample was purified in an ammonium bicarbonate buffer which was apparently not completely removed by cyclesof centifugationand rinsing. Crystallinearrays,which includevisible packing anomalies and mobile monomer sized defects, can evidently result from salts and buffers. Such arrays are unlikely to form when small ions of a single sign of charge are used as counterionsto charged groups of large immobile molecules, simply because repulsive interactions between mobile ions do not favor condensation. We have never observed such images in more highly purified preparations. Unfortunately, these data bear a strong resemblance to earlier work which linked similar appearingislandswith prospects for high-speed DNA sequencing. (35) Stewart, M.; McLachlan, A. D.Nature 1976,257,331. (36) Phillips, G. N., Jr.; Fillers, J. P.; Cohen, C. Biophys. J. 1980,32, 485. Chacko, S.; Phillips,G. N.Biophys. J. 1992,61,1256. (37) Rowe, A. J. h o c . R. SOC.London, B 1964,160,437. (38) Caspar, D. L. D.; Cohen, C.; Longley, W. J. Mol. Biol. 1969,41, 87.
(39) Stewart, M. J. Mol. Biol. 1981,148,411.
3486 Langmuir, Vol. 9, No.12, 1993
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Figure 7. (a) A wide scan image of several molecules of tropomyosin. (b) At higher magnifications, somewhat better resolution is noticeable in regions where chains are nonoverlapping. Vt= 0.5 V for (a, b). (c) STM image of a deposit from an ammonium bicarbonate buffer containing tropomyosin. Many such islands were seen scattered over the surface. (d) Magnified view of (c) showing periodic arrays comprised of ordered monomer-sized units, as judged by roundness and vacancies. Vt = 0.5 V, bar = 200 A.
We have made one attempt to test whether disulfide linkages could be inferred for the "circular" form of proins~1in.l~In this case, however, extreme aggregation phenomena essentiallyprohibits distinguishingindividual molecules, as exemplified in Figure 8a. These strong coverage inhomogeneitieswere again accompanied by foam formation during purging. It remains difficult to believe that these molecules can diffuse on the wet Pt(ll1)surface, so that extreme surfactant-related lateral aggregation seems likely. Again the apparent height in the STM is comparableto the height of a few atomic steps. For many of the molecules described so far, this may not be
I
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Figure8. (a) A wide scan image showingstriking concentration
variations for deposition of recombinant proinsulin. Vt = -0.8 V. (b) A unique image obtained after depositing T-shaped IgG molecules. (c) In the subsequent run,the IgG concentration was increased 10-fold. The bare patch at the step edge is a graphitic layer. Bias voltage of 0.5-1.0V were used for these and remaining molecules as usually only coarse structure could reliably be observed. Bar = loo0 A.
completely unreasonable because it is possible that these molecules may be flattened by strong denaturing interactions with the Pt(ll1)surface. Since some have stated that tunneling could give reasonable topographic heights
STM of Biological Molecules
by virtue of unknown conductivity anomalies, we have attempted to test this possibility by studying a more 3-D protein, bovine immunoglobinG (IgG). An image obtained for this 100 A thick, 300 A wide, T-shaped molecule4 is shown in Figure 8b. Since this was the only region where such T-shaped features were observed during this run, making the data statistically insignificant, the solution concentration was increased by a factor of 10. The resulting sample showed a more uniform distribution of objects which are significantly less T-shaped than the preceding data. One finds that in all these images the contrast appears to saturate at nominal 5-A heights, but 2-D denaturation seems unlikely for this densely interwoven, disulfide bridge stabilizedstructure. Also apparent in this image is a circular island at a step edge which appears free of adsorbates. These islands are comprised of a "graphitic" layer which forms upon annealing from carbon atoms which are not removed during the Pt leaning.^' These islands are observed in every run and they do not bind any of the molecules we have studied. These graphitic patches also provide a height reference which allows us to measure height when the Pt surface is completely saturated by applying IgG at high doses. Experimentally, a maximum 5-A height is still measured but it becomes difficult to maintain tip stability for even a single frame. We thus find no evidence for conductivity anomalies which would allow observation of real topographic heights for truly 3-D molecules. Since a tunnel gap is typically 10A and the tip retracts 5 A a t saturation, contact is imminent for molecules which are more than 15 A thick on the surface. Presumably this relates to the necessity of strong surface bonding for obtaining reproducible data.
Deoxyribonucleic Acids Much of the interest in STM of biological molecules is associated with early results on DNA. Figure 9a shows two overlapping wide scan images of a portion of a double stranded plasmid DNA, pBR322, which contains about 4k base pairs. There is considerable debris, which is expected for Tris-EDTA buffers which are present at 10% by weight levels, but the long, gently curving molecules are quite distinctive. The roughness of this Pt(ll1) surface, which could be improved with better orientation, is clearly problematic for maintaining adequate contrast for such enormous molecules. Figure 9b shows another molecule in a flatter region and one finds a slight apparent height increasewhere the presumablysupercoiledmolecule appears to overlap itself, but the maximum height is still about 5 A. Apparent high resolution internal structure was occasionallyobserved as shown in Figure 9c. Although portions of this image might be interpreted as helical, this is not globally true and these images are not uniquely reproducible. It appears more likely, given the small apparent height and the multiple imaging associated with features separate from the chain, that this internal structure reflects untrustworthy mechanical interactions rather than revealing the pitch of DNA. In a reasonably conservative view of high-resolution images, as in Figure 9d, it appears that these molecules are about 50 A wide and show unreliable internal structure which is likely artifactual. One can select specificregions of such images which show a helical pitch, but nothing is learned from such tautologies. A comparison with TEM work on metal-
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(40)Silverton,E.W.;Navia, M.A; Davies, D.R.Proc. Natl. Acod. Sci.
Figure 9. (a, b) Images of double-stranded plasmid DNA. (c) Apparent internal structure was observed occasionally,. and regions of apparent helicity could be selected. (d) At hgher magnificationsa 50 Awide chain with unreliableinternalstructure could be consistently obtained. Bar = 200 A.
1977, 74,5140. (41) Hallmark, V.M.;Chiang, S.;Wisll, Ch.J. Vac. Sci. Technol.1991, B9, 1111.
shadowed preparations shows that there are still some interesting problems which do not require atomic reso-
Wilson et at.
--* !.
Figure 10. (a) A wide scan image of a nearly circular molecule of single-stranded DNA. A clean graphitic region is seen near the top. (b) A higher magnification image of a different molecule reveals a more extended thread which extends beyond the frame
boundaries. (c) A higher magnification view of a portion of the chain near the center of (b) shows variations in apparent height and sharpness. (d) A t still higher magnifications, one can find regions where the resolution begins to approach the size of monomers but fine structure still suggests complex secondary structure. Images a-c were obtained with V, = 1.0 V, bar = 200 A.
lution$%abut the advantages of STM for such observations are at best arguable. Single-stranded DNA has perhaps aroused the most interest, because of claims for sequencing and because attempts to manipulate single molecules might lead to replicatable structures. Figure 10a shows a wide scan image of a nearly circular molecule of a circular M13 viral (+) single strand situated near patches of graphitic impurities. We find that these molecules rarely appear circular, but instead form regions of dense clumps separated by more extended chains as in Figure lob. For more extended regions, a relatively narrow thread with a zigzag structure can be seen, but the width of the thread is variable, Figure lOc, and the chain cannot be followed in many regions where the DNA appears tangled, or possibly encrusted by the 30% by weight of Tris-EDTA buffers in this sample. Tangled secondarystructure must be expected because of the hydrophobic and pairing character of the bases, as known from TEM studies which use formamide solutions to diminish secondary structure by competing for hydrogen bonding.43 Figure 10d shows a higher magnificationimage of a short, relatively straight segment where internal structure is again visible on a 20-A scale. Even if the resolution can be improved, it is clear that secondarystructure on the scaleof tens of bases would severely complicate attempts at STM-based sequencing. (42) Bortner, C.; Grifith, J. J. Mol. Biol. 1990,225,623. (43) Heieh, C.; Griffith, J. D.h o c . Natl. Acad. Sci. 1989,86,4833.
Experimental Artifacts In the course of these experiments we have acquired much experience with artifacts which appear to be associated with the build up of molecular fragments on the tip. For example, Figure l l a shows how a deleterious tip change can transform an image of isolated debris into an image which appears to show structured chains. If the tip is dull and sharp asperities are well separated, then one can observe images such as Figure l l b which show repeated shapes which can be altered by modifying the tip. Such data must be rejected unless orientational analogs are found, irregardless of resemblances to the desired results. Experienced operators routinely use such images to interactively obtain and verify tip sharpness. Figure llc,d shows a pair of images obtained by reversing the scan direction. On inspection one finds three regions where the contrast also reverses. These regions have the typical appearanceof graphiticdepressions,and we suggest these effects are due to mechanical effects of the tip on adsorbates. Figure l l e shows an image where the horizontal scan is reversed at the image center. The doublestranded DNA molecule, which was visible in both halves of the image at the top of the frame, nearly disappears in the right image after a deleterioustip change. A qualitative consideration of the image contrast saturation suggests a rather simplemodel which might explain such phenomena. We suggest that the presence of molecular adsorbates allows metallic conduction electrons, which generally prefer to be delocalized,to slightlyincrease their extension
STM of Biological Molecules
Langmuir, Vol. 9, No. 12, 1993 3489
is possible for adsorbate-covered tips. If the molecular contrast is saturated by tip-bound adsorbates which essentially fill the tunnel gap, then one might expect that surface adsorbed moleculeswill simply be invisible. Under these conditions, mechanical interactions are likely and one can get into a situation where the tip-bound adsorbates are forced out of or into the gap when scanning in opposite directions, leading to direction-dependent contrast. Such mechanical interactions may well result in irreversiblescan induced damage to molecules. Such an event occurred during acquisition of Figure 9b, where the strands disappear near the bottom of the frame. These portions of the molecule appeared normal in the preceding frame but appeared permanently damaged in subsequent images taken after the tip was restored. These contact phenomena are accompanied by noisy image characteristics and we have learned to restore the tip and examine noise levels and distortions at Pt(ll1) step edges before searching for another molecule. These effects are not very problematic for small or thin molecules, but become a serious problem for double stranded DNA where the tip functions properly about half of the time. For molecules like IgG, we find that tips are slimed within an hour, and the majority of time is spent installing replacements or attempting to restore tip function.
i
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I Figure 11. (a, b) Artifactchains resultingfrom blunt tips. (ee) Images showing scan direction dependent contrast associated with mechanical interactions. Bar = 500 A.
into the vacuum by admixing with molecular states localized at the interface. By reciprocity, a similar effect
Conclusions This body of data allows us to draw several conclusions. First, STM renders credible high resolution images for small, thin molecules provided adequate surface binding is involved. Unfortunately, it is likely that strong molecule-surface interactions which result in unique conformations for monomers will not establish a unique or static conformationfor flexiblepolymers. For sufficientlysimple oligomers one could attempt to identify the observed conformations in terms of models which include surfaceinduced molecular distortions, possibly attempting to work from monomers to dimers to trimers, etc. Alternatively, one may choose to regard conformational complexity as a phenomenological blurring which allows the detection of the molecule but obscures details over distances comparable to the lengths of the side chains. For large polymers, extensive blurring is expected for overlapping or 3-D structures because of limitations on tunneling processes for insulating molecules. This leads to tipmolecule contact which results in a variety of artifacts for molecules which exceed about 10 in thickness. In some cases it might be preferable to use a weakly binding substrate in order to prevent denaturing and allow ordering. However, for complex polymers one cannot expect crystallization but rather must anticipate even worse problems with tip contact interactions with thick undenatured molecules. It is unfortunate that we can obtain data similar to that of several highly optimistic publications if we overlook impurities, statistical significance, and sensible physical mechanisms. It is also unfortunate that such data have served as the justification for the proposed development of new sequencing techniques, including STM variants employing near field optical probes and magnetic resonance detection. Although we applaud novel research in any important new area, these variants often involve a serious degradation of lateral resolution relative to STM, because of the long range character of the forces and optical decay lengths involved,which have not been addressed, and often assume that simple molecular conformations can be easily obtained. Although such techniques may find a viable niche, they seem unlikely to succeed at obtaining high-resolution objectives in the context of biological molecules. Indeed,
3490 Langmuir, Vol.9,No. 12, 1993 the inadequate lateral resolution in our images of DNA is already significantly better than that reported for AFM images. Our extensive efforts have taught us to greatly appreciate the structural insensitivity and easy statistics of chemical sequencing techniques and of the 3-D sensitivity and averaging capabilities of techniques like solution NMR. There are several aspects of the present work which might be fruitfully extended. For rigid planar molecules it seems likely that a great deal may be learned about molecular binding and intermolecular interactions on surfaces. Surfaces dressed with molecules with specific functional properties are already useful in many contexts. High resolution images of uncrystallized, unstained materials such as CaM are not to be found in the literature and may be of value in specific cases, such as posttranscriptional modifications,which are beyond our knowledge. The low concentrations and lack of salts and buffers in the present work can be modified by using brief exposures and rinses in a thin-layer flow cell. Under theseconditions, extensions to currently interesting homopolymer aggregation and transient denaturation phenomena might be po~sible.~ Alternatively, *~~ one could attempt to shrink the lateral dimensions of a droplet to a few micrometers, thereby gaining access to studies requiring only a few molecules. These data also provide some insight into molecular aggregationphenomena which may be important in a number of contexts. For example, there is much current interest in the aggregation of short amylloid peptides which are linked to Alzheimers disease.For this important problem there are many competing techniques with different strengths and disadvantages, but most methods face extreme difficulty when faced with variably sized aggregates of molecules which can take on multiple conformations. Although the information obtained from STM will provide little insight on the atomic scale, sufficiently thin aggregates can be examined one by one. With AFM one might well obtain a full height representation and avoid some of the contact problems, but poor lateral resolution would still be expected. Alternatively, one might abandon these microscopy techniques altogether and consider the use of the Pt(ll1) surface, possibly largely passivated by small molecules like hydroquinone, to serve as a universal binder for molecules like IgG which could then selectively form complexes with specific target molecules.
Prospects In a longer view, it appears that for 3-D molecules one will wish to employ techniques which operate at the largest (44) Ismail, A. A.; Mantach, H. H. Biopolymers 1992,32,1181. Bello, J. Biopolymers 1992, 32, 185. (46) Mo, J.; Holtzer, M. E.; Holtzer, A. Biopolymers 1991, 31, 1417. (46) Zagorski, M. G.; Barrow, C. J. Biochemistry 1992, 31, 5621. (47) Caputo, C. B.; Sobel, I. R. E.; Scott, C. W.; Brunner, W. F.; Barth, P. T.; Blowers, D. P. Biochem. Biophys. Res. Commun. 1992,185,1034. (48) Burdick, D.; Soreghan, B.; Kwon, M.; Kosmoski, J.; Knauer, M.; Henachen, A.; Yates, J.; Cotman, C.; Glabe, C. J. Biol. Chem. 1992,267, 546.
Wilson et al.
possible tip-sample spacings sufficient to coarsely detect the objects of interest. The emphasis in this case should shift from elegant imaging to clever experiments where one might, for example, develop reaction cells wherein terminally anchored molecules can be imaged and subsequently solvated and reacted with binding proteins and activators and the resulting complexes imaged again. The advance here is not one where the microscopy cannot be done any other way, but rather that the technique becomes rapid and reliable enough to be of more value than the numerous alternatives. This perspective is reflected in our work on metal surfaces where we initially carried out experimentally difficult, somewhat myopic, studies of wellknown atomic g e ~ m e t r i e sand ~ ~ ultimately learned that easier measurements at much larger length scales can provide unique insights into previously unknown ordering and nucleation and growth phenomena.m Finally, we would not rule out the possibility that advances might ultimately lead to a viableDNA sequencing technique. For example, optical techniques now exist which can help to nearly linearize macroscopic DNA molecules, hopefully allowing elimination of much of the complexity inherent in the circular molecules described here. It is rather unlikely that textbook structures, with all nucleotides equivalent, will be formed to allow easy identification of the subunits. However, the attained resolution might be adequate to deduce the order of bases if some distinctive, conformation-independent signature was available. Presently there are no such techniques known for STM because the conduction electrons involved are highly sensitive to the local environment as well as to atomic identities. Combinations of STM detection with frequency-selective excitations might help to solve this problem, but we do not know whether such advances are feasible or whether they might be sufficiently fast,reliable, or accurate to compete with other rapidly evolving technologies. It is our belief that continued work in scanning probe microscopies will ultimately contribute to improved understanding of some aspect of biochemistry, provided broadly knowledgeable experts are equipped with reliable instrumentation, reasonable expectations, and accurate interpretive skills. It is our hope that these results might be of some assistance in identifying potentially soluble problems and in motivating new ideas and more diligent experimental efforts.
Acknowledgment. We are pleased to acknowledge helpful interactions with patient colleagues in many disciplines who are, unfortunately, too numerous to list. (49) Hallmark, V. M.; Chiang, S.; Rabolt,J. F.; Swalen, J. D.; Wileon, R. J. Phys. Reu. Lett. 1987, 59,2879. (50) Chambliss, D. D.; Wilson, R. J.; Chiang, S.Phys. Rev. Lett. 1991, 66. 1721.
