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Comparison of scanning tunnelling microscopy and transmission electron microscopy image data of a microbial polysaccharide
Ultramicroscopy48 (1993) 197-201 North-Holland
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Comparison of scanning tunnelling microscopy and transmission electron microscopy image data of a microbial polysaccharide M.J. Wilkins a, M.C. D a v i e s a, D . E . J a c k s o n a, J.R. M i t c h e l l b, C.J. R o b e r t s a, B.T. S t o k k e c a n d S.J.B. T e n d l e r a a The VG STM Laboratory for Biological Applications, Department of Pharmaceutical Sciences, The University of Nottingham, University Park, Nottingham NG7 2RD, UK b Department of Applied Biochemistry and Food Science, The University of Nottingham, Sutton Bonnington, Loughborough, Leicestershire LE12 5RD, UK c Norwegian Biopolymer Laboratory, Department of Physics, University of Trondheim, NTH, N-7034 Trondheim, Norway
Received 24 June 1992
The study of biological material by scanning tunnelling microscopyhas led to a need for critical validation of the resultant data. We present here a comparison of scanning tunnelling microscopy images and electron micrographs of a microbial polysaccharide. Xanthan gum in a glycerol solution was sprayed onto mica discs and coated with a platinum/carbon conducting layer and a carbon backing layer. The ability of the scanning tunnelling microscopeto image replicas as prepared for electron microscopyis demonstrated. The images of isolated xanthan molecules have comparable contrast to electron micrographs and in addition, contain directly available 3D data. The implications of these results, for future progress in sample preparation for STM studies, are discussed. Further images of xanthan, coated only with platinum/carbon, are used to illustrate that the resolution can be improved by a reduction in the grain size of the coating.
1. Introduction It is becoming widely acknowledged that the application of scanning tunnelling microscopy (STM) to the study of biomolecules [1-3] is not as facile as early studies had suggested. The technique has suffered a number of problems which include image interpretation [4], the presence of substrate features which resemble biomolecules [5,6] and the lack of reproducibility when imaging biological material [7]. These problems indicate the need for data validation by the comparison of STM results with those from complementary microscopy techniques. In this study we have examined a microbial polysaccharide, xanthan gum, using both electron microscopy (EM) and STM.
The xanthan samples for both microscopy techniques were prepared using spray deposition followed by metal coating, methods that have been commonly utilized for E M sample preparation of biological macromolecules [8]. Unfortunately, preliminary studies on uncoated xanthan failed to reveal any molecular information. This is probably due to tip-induced movement of the xanthan molecules outside the scan area of the STM tip. Typically, following deposition in a 60% glycerol solution and vacuum drying, the samples were coated with evaporated p l a t i n u m / c a r b o n ( P t / C ) and imaged with E M or STM. This metal coating overcomes problems associated with STM tip-induced sweeping [9-11], allowing the direct imaging of the coated molecules [12-15]. The
0304-3991/93/$06.00 © 1993 - Elsevier Science Publishers B.V. All rights reserved
198
M.J. 14~lkinset al. / STM and TEM of microbialpolysaccharide
established literature on E M of xanthan allows the assessment and direct correlation with STM data.
2. Experimental details 2.1. Sample preparation 2.1.1. Platinum/carbon and carbon-coated samples The xanthan samples were prepared by purifying solutions of xanthan by ultracentrifugation, and fragmented by sonication as previously described [16]. The samples were dissolved in 0.1M ammonium acetate buffer at a concentration of approximately 1 m g / m l . The solutions were further diluted to a working concentration of 80 /~g/ml with 60% glycerol. Aliquots of the xanthan solution in glycerol (50 /zl) were sprayed onto freshly cleaved mica discs and dried at approximately 10-6 Torr for 1 hour. The samples were rotary-shadowed with a 0.6 to 0.7 nm thick P t / C (95% : 5%) layer at an incident angle of 5 °, followed by a 6 to 7 nm thick carbon backing layer at an incident angle of 90 °. These replicas were transferred to copper grids and highly oriented pyrolytic graphite ( H O P G ) substrates and examined by EM and STM, respectively. 2.1.2. Platinum / carbon-coated samples Further replicas of non-fragmented xanthan were also produced by spraying xanthan (10 t z g / m l in deionized water) onto freshly cleaved mica discs and drying at 10 -5 Torr. The samples were rotary-shadowed with a P t / C (95% :5%) layer, at an incident angle of 45 °. These replicas were imaged by the STM directly on the mica substrate. 2.2. Sample analysis Z2.1. S T M The samples were scanned under ambient conditions using a V G STM 2000 (VG Microtech, Uckfield, UK) employing a negatively biased, mechanically cut p l a t i n u m / i r i d i u m ( 8 0 % : 2 0 % ) tip.
Fig. 1. Electron micrograph of sonicated xanthan. The xanthan was sprayed onto a mica disc, rotary-shadowedwith a Pt/C layer and a carbon backing layer, and the replica transferred to a copper grid for examination by EM. Scale bar = 200 nm. The microscope was operated in the constantcurrent mode, with the tunnelling current and voltage being set at 30 pA and 1.5 V, respectively. 2.2.2. E M The electron micrographs were obtained from the replicas employing a Philips E M 400T electron microscope operated in transmission mode at 80 keV accelerating voltage with a 50 /~m aperture and nominal electron optic magnification between 17000 x and 46000 × . The magnification was calibrated using a grid of 1200 lines/ram ($102, Agar Aids).
3. Results The image in fig. 1 is representative of the electron micrographs taken of the sonicated sam-
M.J. geilkins et al. / STM and TEM of microbial polysaccharide
199
~iiii:i !! ilii ii
Fig. 2. 1385 n m × 1385 nm STM topograph of sonicated xanthan molecules, sprayed onto a mica substrate and rotaryshadowed with a P t / C layer and carbon backing layer. The replica was transferred to an HOPG substrate prior to scanning with the STM. The image shows various molecular weight species of xanthan molecules distributed across the substrate. Scale bar = 100 rim. pies, showing a number of entangled xanthan molecules of various lengths, indicative of a polydisperse polymer population. The average diame-
Fig. 3. Higher-magnification 793 n m × 793 nm STM topograph showing individual xanthan molecules from the area depicted in fig. 2. The grain diameter of the coating and xanthan molecules ranges from 16 to 20 nm. Scale bar = 100 nm.
t e r o f t h e x a n t h a n m o l e c u l e s r a n g e s f r o m 9 t o 10 nm. T h e i m a g e s i n figs. 2 a n d 3 a r e S T M t o pographs of the Pt/C and carbon-coated xanthan
Fig. 4. 996 n m × 591 nm scan of xanthan sprayed onto a mica disc and rotary-shadowed with a P t / C layer. The xanthan molecule was imaged directly on the mica substrate and has a maximum diameter of 8 nm. The molecule forms two loops suggesting the presence of single- and double- strand conformations. Scale bar = 100 nm.
200
M.J. Wilkins et al. / STM and TEM of microbial polysaccharide
in the presence of observed artefacts compared to naked HOPG substrates. In our experience, the artefactual features that are present in the replicas appear as straight lines, of high contrast, and are readily distinguished from biological material.
4. Discussion
Fig. 5. 389 nm x 389 nm scan of xanthan coated with a P t / C layer. The average grain diameter of the coating is 5 nm and of the individual xanthan molecule is 8 nm. Scale bar = 100 nm.
molecules. Fig. 2 displays a 1385 nm × 1385 nm scan showing several convoluted and linear macromolecules distributed across the substrate surface, and fig. 3 shows a higher-resolution scan of individual molecules from this area. The grain size of the carbon backing layer ranges from 16 to 20 nm in diameter which corresponds to the observed width of the xanthan molecules. Again, the polydisperse polymer population is dearly evident. Figs. 4 and 5 show high-resolution STM images of the single unsonicated xanthan molecules, coated only with a P t / C layer. Again, the xanthan molecules were well distributed across the substrate and display their entangled polydisperse nature. The average grain size of the P t / C coating is 5 nm and the average diameter of the xanthan molecules is 8 nm. Interestingly, the dimensions are similar to those of xanthan molecules in previous EM studies [16,17]. A xanthan molecule, shown in fig. 4, separates from a double strand into single strands, forming two loops, consistent with the conformations reported in EM studies [17]. In addition to the immobilization effect, the evaporation replicas show a significant reduction
These results demonstrate the potential value of EM techniques for preparing biological samples for STM imaging. The merit of using spraying as a deposition method to produce images of single xanthan molecules is evident. The presence of individual molecules distributed across the substrate is also indicative of the uniform coverage of the mica surface. In practice this reduces the time for location of biomolecules and is supported by our immediate identification of biological material upon scanning new areas of a replica. Furthermore, the use of conductive coatings has proven to be a suitable method of sample immobilization as demonstrated here and in previous studies of recA-DNA complexes [12,13], T7 bacteriophage [14] and tobacco mosiac virus [15]. The mechanical strength of the electron-beamevaporated P t / C and carbon layers counteracts the sweeping effect of the tip on the biomolecules. The thin P t / C layer alone is also resistant to deformation by the scanning tip when imaged under ambient conditions, contrasting with other metallic coatings which can exhibit mobility on the surface [9]. In addition to overcoming some of the problems associated with biological STM, the EM preparation techniques have allowed a direct comparison between electron micrographs and STM topographs. The correlation between these images is excellent, validating the data obtained from the STM. Following this protocol, information on molecular size, shape and possibly conformation, obtained by STM, has been confirmed by EM and thus represents an important preliminary step in the study of any novel biological material by STM. The improved resolution yielded by the P t / C film compared to the larger-grained carbon film
M.Z Wilkins et al. / STM and TEM of microbial polysaccharide
indicates the significance of the relationship between grain size and molecular dimension. Further developments in high-resolution shadowing techniques [18] should reveal greater molecular detail. The presence of anomalous artefactual features in STM images of surfaces has been attributed to a convolution effect between crystal planes in the sample and the imaging tip [19,20], or the presence of grain boundaries [21]. Therefore, the significant reduction of artefacts observed on the evaporated replicas can be accounted for by the non-crystalline surface produced by the evaporation process. 5. Conclusion In the studies detailed within this communication, we have explored the application of EM coating and spraying technology to biological STM. The images of the xanthan samples have shown excellent correlation between STM and EM. In addition the coating procedure overcomes the immobilization problems, prevalent in studies of naked biomolecules by STM. The spray deposition method has allowed us to image individual xanthan molecules uniformly distributed over the substrate surface. Acknowledgements We wish to thank the M A F F / D T I hydration (HYDRA) Link programme for their continued support in these studies. We would also like to acknowledge the SERC Protein Engineering/DTI Link programme, Glaxo Research Ltd. and VG Microtech for their support. References [1] R.D. Edstrom, X. Yang, G. Lee and D.F. Evans, FASEB 4 (1990) 3144.
201
[2] M.G. Youngquist, R.J. Driscoll, T.R. Coley, W.A. Goddard and J.D. Baldeschwieler, J. Vac. Sci. Technol. B 9 (1991) 1304. [3] A. Cricenti, S. Selei, A.C. Felici, R. Generosi, E. Gori, W. Djaczenko and G. Chiarotti, Science 245 (1989) 1226. [4] S.M. Lindsay, T. Thundat and L. Nagahara, J. Microscopy 152 (1988) 213. [5] C.R. Clemmer and T.P. Beebe, Jr., Science 251 (1991) 640. [6] M.C. Davies, D.E. Jackson, K.M. Kreusel, C.J. Roberts, S.J.B. Tendler, M.J. Wilkins and P.M. Williams, Polymer Surfaces II Scanning Probe Microscopy: Current Issues in the Analysis of Polymeric Biomolecules, Ed. J. Feast (Wiley, New York, in press). [7] A. Stemmer, A. Hefti, U. Aebi and A. Engel, Ultramicroscopy 30 (1989) 263. [8] J.M. Tyler and D. Branton, J. Ultrastruct. Res. 71 (1980) 95. [9] C.J. Roberts, M.J. Wilkins, M.C. Davies, D.E. Jackson and S.J.B. Tendler, Surf. Sci. Lett. 261 (1992) L29. [10] C.J. Roberts, M. Sekowski, M.C. Davies, D.E. Jackson, M.R. Price and S.J.B. Tendler, Biochem. J. 280 (1992) 181. [11] T.E. Wilson, M.N. Murray, D.F. Ogletree, M.D. Bednarski, C.R. Cantor and M.B. Salmeron, J. Vac. Sci. Technol. B 9 (1991) 1171. [12] G. Travaglini, E. Stoll, M. Amrein, A. Stasiak and H. Gross, J. Microsc. Spectrosc. Electron. 13 (1988) 257. [13] M. Amrein, A. Stasiak, H. Gross, E. Stoll and G. Travaglini, Science 240 (1988) 514. [14] R. Garcia, D. Keller, J. Panitz, D.G. Bear and C. Bustamante, Ultramicroscopy 27 (1989) 367. [15] J.G. Mantovani, D.P. Allison, W.J. Warmack, T.L. Fertell, J.R. Ford, R.E. Manos, J.R. Thompson, B.B. Reddick and K. Bruce Jacobson, J. Microscopy 158 (1990) 109. [16] B.T.Stokke, A. EIgsaeter and O. Smidsrcd, Electron Microscopy of Xanthan Topology and Strandedness of the Ordered and Disordered Conformation, ACS Symp. Ser. 396 (1989) 145. [17] B.T. Stokke, O. Smidsrcd and A. Elgsaeter, Biopolymers 28 (1989) 617. [18] R. Wepf, M. Amrein, U. Burkli and H. Gross, J. Micrcoscopy 163 (1991) 51. [19] P.I. Oden, T. Thundat, L.A. Nagahara, S.M. Lindsay, G.B. Adams and O.F. Sankey, unpublished. [20] M.J. Wilkins, M.C. Davies, D.E. Jackson, C.J. Roberts, S.J.B. Tendler and P.M. Williams, Appl. Phys. Lett. 60 (1992) 1436. [21] W. Heckl and G. Binnig, in: Proc. STM '91, Ultramicroscopy 42-44 (1992) 1073.
rn~mJi,
nntl~n
Comparison of scanning tunnelling microscopy and transmission electron microscopy image data of a microbial polysaccharide M.J. Wilkins a, M.C. D a v i e s a, D . E . J a c k s o n a, J.R. M i t c h e l l b, C.J. R o b e r t s a, B.T. S t o k k e c a n d S.J.B. T e n d l e r a a The VG STM Laboratory for Biological Applications, Department of Pharmaceutical Sciences, The University of Nottingham, University Park, Nottingham NG7 2RD, UK b Department of Applied Biochemistry and Food Science, The University of Nottingham, Sutton Bonnington, Loughborough, Leicestershire LE12 5RD, UK c Norwegian Biopolymer Laboratory, Department of Physics, University of Trondheim, NTH, N-7034 Trondheim, Norway
Received 24 June 1992
The study of biological material by scanning tunnelling microscopyhas led to a need for critical validation of the resultant data. We present here a comparison of scanning tunnelling microscopy images and electron micrographs of a microbial polysaccharide. Xanthan gum in a glycerol solution was sprayed onto mica discs and coated with a platinum/carbon conducting layer and a carbon backing layer. The ability of the scanning tunnelling microscopeto image replicas as prepared for electron microscopyis demonstrated. The images of isolated xanthan molecules have comparable contrast to electron micrographs and in addition, contain directly available 3D data. The implications of these results, for future progress in sample preparation for STM studies, are discussed. Further images of xanthan, coated only with platinum/carbon, are used to illustrate that the resolution can be improved by a reduction in the grain size of the coating.
1. Introduction It is becoming widely acknowledged that the application of scanning tunnelling microscopy (STM) to the study of biomolecules [1-3] is not as facile as early studies had suggested. The technique has suffered a number of problems which include image interpretation [4], the presence of substrate features which resemble biomolecules [5,6] and the lack of reproducibility when imaging biological material [7]. These problems indicate the need for data validation by the comparison of STM results with those from complementary microscopy techniques. In this study we have examined a microbial polysaccharide, xanthan gum, using both electron microscopy (EM) and STM.
The xanthan samples for both microscopy techniques were prepared using spray deposition followed by metal coating, methods that have been commonly utilized for E M sample preparation of biological macromolecules [8]. Unfortunately, preliminary studies on uncoated xanthan failed to reveal any molecular information. This is probably due to tip-induced movement of the xanthan molecules outside the scan area of the STM tip. Typically, following deposition in a 60% glycerol solution and vacuum drying, the samples were coated with evaporated p l a t i n u m / c a r b o n ( P t / C ) and imaged with E M or STM. This metal coating overcomes problems associated with STM tip-induced sweeping [9-11], allowing the direct imaging of the coated molecules [12-15]. The
0304-3991/93/$06.00 © 1993 - Elsevier Science Publishers B.V. All rights reserved
198
M.J. 14~lkinset al. / STM and TEM of microbialpolysaccharide
established literature on E M of xanthan allows the assessment and direct correlation with STM data.
2. Experimental details 2.1. Sample preparation 2.1.1. Platinum/carbon and carbon-coated samples The xanthan samples were prepared by purifying solutions of xanthan by ultracentrifugation, and fragmented by sonication as previously described [16]. The samples were dissolved in 0.1M ammonium acetate buffer at a concentration of approximately 1 m g / m l . The solutions were further diluted to a working concentration of 80 /~g/ml with 60% glycerol. Aliquots of the xanthan solution in glycerol (50 /zl) were sprayed onto freshly cleaved mica discs and dried at approximately 10-6 Torr for 1 hour. The samples were rotary-shadowed with a 0.6 to 0.7 nm thick P t / C (95% : 5%) layer at an incident angle of 5 °, followed by a 6 to 7 nm thick carbon backing layer at an incident angle of 90 °. These replicas were transferred to copper grids and highly oriented pyrolytic graphite ( H O P G ) substrates and examined by EM and STM, respectively. 2.1.2. Platinum / carbon-coated samples Further replicas of non-fragmented xanthan were also produced by spraying xanthan (10 t z g / m l in deionized water) onto freshly cleaved mica discs and drying at 10 -5 Torr. The samples were rotary-shadowed with a P t / C (95% :5%) layer, at an incident angle of 45 °. These replicas were imaged by the STM directly on the mica substrate. 2.2. Sample analysis Z2.1. S T M The samples were scanned under ambient conditions using a V G STM 2000 (VG Microtech, Uckfield, UK) employing a negatively biased, mechanically cut p l a t i n u m / i r i d i u m ( 8 0 % : 2 0 % ) tip.
Fig. 1. Electron micrograph of sonicated xanthan. The xanthan was sprayed onto a mica disc, rotary-shadowedwith a Pt/C layer and a carbon backing layer, and the replica transferred to a copper grid for examination by EM. Scale bar = 200 nm. The microscope was operated in the constantcurrent mode, with the tunnelling current and voltage being set at 30 pA and 1.5 V, respectively. 2.2.2. E M The electron micrographs were obtained from the replicas employing a Philips E M 400T electron microscope operated in transmission mode at 80 keV accelerating voltage with a 50 /~m aperture and nominal electron optic magnification between 17000 x and 46000 × . The magnification was calibrated using a grid of 1200 lines/ram ($102, Agar Aids).
3. Results The image in fig. 1 is representative of the electron micrographs taken of the sonicated sam-
M.J. geilkins et al. / STM and TEM of microbial polysaccharide
199
~iiii:i !! ilii ii
Fig. 2. 1385 n m × 1385 nm STM topograph of sonicated xanthan molecules, sprayed onto a mica substrate and rotaryshadowed with a P t / C layer and carbon backing layer. The replica was transferred to an HOPG substrate prior to scanning with the STM. The image shows various molecular weight species of xanthan molecules distributed across the substrate. Scale bar = 100 rim. pies, showing a number of entangled xanthan molecules of various lengths, indicative of a polydisperse polymer population. The average diame-
Fig. 3. Higher-magnification 793 n m × 793 nm STM topograph showing individual xanthan molecules from the area depicted in fig. 2. The grain diameter of the coating and xanthan molecules ranges from 16 to 20 nm. Scale bar = 100 nm.
t e r o f t h e x a n t h a n m o l e c u l e s r a n g e s f r o m 9 t o 10 nm. T h e i m a g e s i n figs. 2 a n d 3 a r e S T M t o pographs of the Pt/C and carbon-coated xanthan
Fig. 4. 996 n m × 591 nm scan of xanthan sprayed onto a mica disc and rotary-shadowed with a P t / C layer. The xanthan molecule was imaged directly on the mica substrate and has a maximum diameter of 8 nm. The molecule forms two loops suggesting the presence of single- and double- strand conformations. Scale bar = 100 nm.
200
M.J. Wilkins et al. / STM and TEM of microbial polysaccharide
in the presence of observed artefacts compared to naked HOPG substrates. In our experience, the artefactual features that are present in the replicas appear as straight lines, of high contrast, and are readily distinguished from biological material.
4. Discussion
Fig. 5. 389 nm x 389 nm scan of xanthan coated with a P t / C layer. The average grain diameter of the coating is 5 nm and of the individual xanthan molecule is 8 nm. Scale bar = 100 nm.
molecules. Fig. 2 displays a 1385 nm × 1385 nm scan showing several convoluted and linear macromolecules distributed across the substrate surface, and fig. 3 shows a higher-resolution scan of individual molecules from this area. The grain size of the carbon backing layer ranges from 16 to 20 nm in diameter which corresponds to the observed width of the xanthan molecules. Again, the polydisperse polymer population is dearly evident. Figs. 4 and 5 show high-resolution STM images of the single unsonicated xanthan molecules, coated only with a P t / C layer. Again, the xanthan molecules were well distributed across the substrate and display their entangled polydisperse nature. The average grain size of the P t / C coating is 5 nm and the average diameter of the xanthan molecules is 8 nm. Interestingly, the dimensions are similar to those of xanthan molecules in previous EM studies [16,17]. A xanthan molecule, shown in fig. 4, separates from a double strand into single strands, forming two loops, consistent with the conformations reported in EM studies [17]. In addition to the immobilization effect, the evaporation replicas show a significant reduction
These results demonstrate the potential value of EM techniques for preparing biological samples for STM imaging. The merit of using spraying as a deposition method to produce images of single xanthan molecules is evident. The presence of individual molecules distributed across the substrate is also indicative of the uniform coverage of the mica surface. In practice this reduces the time for location of biomolecules and is supported by our immediate identification of biological material upon scanning new areas of a replica. Furthermore, the use of conductive coatings has proven to be a suitable method of sample immobilization as demonstrated here and in previous studies of recA-DNA complexes [12,13], T7 bacteriophage [14] and tobacco mosiac virus [15]. The mechanical strength of the electron-beamevaporated P t / C and carbon layers counteracts the sweeping effect of the tip on the biomolecules. The thin P t / C layer alone is also resistant to deformation by the scanning tip when imaged under ambient conditions, contrasting with other metallic coatings which can exhibit mobility on the surface [9]. In addition to overcoming some of the problems associated with biological STM, the EM preparation techniques have allowed a direct comparison between electron micrographs and STM topographs. The correlation between these images is excellent, validating the data obtained from the STM. Following this protocol, information on molecular size, shape and possibly conformation, obtained by STM, has been confirmed by EM and thus represents an important preliminary step in the study of any novel biological material by STM. The improved resolution yielded by the P t / C film compared to the larger-grained carbon film
M.Z Wilkins et al. / STM and TEM of microbial polysaccharide
indicates the significance of the relationship between grain size and molecular dimension. Further developments in high-resolution shadowing techniques [18] should reveal greater molecular detail. The presence of anomalous artefactual features in STM images of surfaces has been attributed to a convolution effect between crystal planes in the sample and the imaging tip [19,20], or the presence of grain boundaries [21]. Therefore, the significant reduction of artefacts observed on the evaporated replicas can be accounted for by the non-crystalline surface produced by the evaporation process. 5. Conclusion In the studies detailed within this communication, we have explored the application of EM coating and spraying technology to biological STM. The images of the xanthan samples have shown excellent correlation between STM and EM. In addition the coating procedure overcomes the immobilization problems, prevalent in studies of naked biomolecules by STM. The spray deposition method has allowed us to image individual xanthan molecules uniformly distributed over the substrate surface. Acknowledgements We wish to thank the M A F F / D T I hydration (HYDRA) Link programme for their continued support in these studies. We would also like to acknowledge the SERC Protein Engineering/DTI Link programme, Glaxo Research Ltd. and VG Microtech for their support. References [1] R.D. Edstrom, X. Yang, G. Lee and D.F. Evans, FASEB 4 (1990) 3144.
201
[2] M.G. Youngquist, R.J. Driscoll, T.R. Coley, W.A. Goddard and J.D. Baldeschwieler, J. Vac. Sci. Technol. B 9 (1991) 1304. [3] A. Cricenti, S. Selei, A.C. Felici, R. Generosi, E. Gori, W. Djaczenko and G. Chiarotti, Science 245 (1989) 1226. [4] S.M. Lindsay, T. Thundat and L. Nagahara, J. Microscopy 152 (1988) 213. [5] C.R. Clemmer and T.P. Beebe, Jr., Science 251 (1991) 640. [6] M.C. Davies, D.E. Jackson, K.M. Kreusel, C.J. Roberts, S.J.B. Tendler, M.J. Wilkins and P.M. Williams, Polymer Surfaces II Scanning Probe Microscopy: Current Issues in the Analysis of Polymeric Biomolecules, Ed. J. Feast (Wiley, New York, in press). [7] A. Stemmer, A. Hefti, U. Aebi and A. Engel, Ultramicroscopy 30 (1989) 263. [8] J.M. Tyler and D. Branton, J. Ultrastruct. Res. 71 (1980) 95. [9] C.J. Roberts, M.J. Wilkins, M.C. Davies, D.E. Jackson and S.J.B. Tendler, Surf. Sci. Lett. 261 (1992) L29. [10] C.J. Roberts, M. Sekowski, M.C. Davies, D.E. Jackson, M.R. Price and S.J.B. Tendler, Biochem. J. 280 (1992) 181. [11] T.E. Wilson, M.N. Murray, D.F. Ogletree, M.D. Bednarski, C.R. Cantor and M.B. Salmeron, J. Vac. Sci. Technol. B 9 (1991) 1171. [12] G. Travaglini, E. Stoll, M. Amrein, A. Stasiak and H. Gross, J. Microsc. Spectrosc. Electron. 13 (1988) 257. [13] M. Amrein, A. Stasiak, H. Gross, E. Stoll and G. Travaglini, Science 240 (1988) 514. [14] R. Garcia, D. Keller, J. Panitz, D.G. Bear and C. Bustamante, Ultramicroscopy 27 (1989) 367. [15] J.G. Mantovani, D.P. Allison, W.J. Warmack, T.L. Fertell, J.R. Ford, R.E. Manos, J.R. Thompson, B.B. Reddick and K. Bruce Jacobson, J. Microscopy 158 (1990) 109. [16] B.T.Stokke, A. EIgsaeter and O. Smidsrcd, Electron Microscopy of Xanthan Topology and Strandedness of the Ordered and Disordered Conformation, ACS Symp. Ser. 396 (1989) 145. [17] B.T. Stokke, O. Smidsrcd and A. Elgsaeter, Biopolymers 28 (1989) 617. [18] R. Wepf, M. Amrein, U. Burkli and H. Gross, J. Micrcoscopy 163 (1991) 51. [19] P.I. Oden, T. Thundat, L.A. Nagahara, S.M. Lindsay, G.B. Adams and O.F. Sankey, unpublished. [20] M.J. Wilkins, M.C. Davies, D.E. Jackson, C.J. Roberts, S.J.B. Tendler and P.M. Williams, Appl. Phys. Lett. 60 (1992) 1436. [21] W. Heckl and G. Binnig, in: Proc. STM '91, Ultramicroscopy 42-44 (1992) 1073.