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Artifacts in Adhesion Force Images Obtained by Force Curve Mapping Katsumi Sugisaki*,† and Nobuyuki Nakagiri† Tsukuba Research Laboratory, Nikon Corporation, 5-9-1 Tokodai, Tsukuba, Ibaraki 300-2635, Japan
Yasuhito Kinjo Radiation Laboratories, Tokyo Metropolitan Industrial Technology Research Institute, 2-11-1 Fukazawa, Setagaya-ku, Tokyo 158-0081, Japan Received June 24, 1998. In Final Form: April 27, 1999 We applied force curve mapping to chromosomes. Two types of artifacts were observed in the adhesion force images thus obtained. Location shifting between the adhesion and topographic images and deformation of the measured adhesive structure were seen. These artifacts are explained by theoretical analysis to be the result of cantilever tip sliding due to cantilever deflection during the adhesion force measurements. Analysis also showed that this tip sliding caused a small, highly adhesive region to escape measurement. The artifacts observed are not small enough to be negligible for molecular-size applications, and therefore, possible solutions to eliminate them are discussed.
I. Introduction Adhesive properties have been derived from atomic force microscopy (AFM)1 for application in various fields of research. Since such adhesive properties are determined by the interaction between the AFM probe tip and the sample surface, it is possible to characterize the sample surface by using a known probe tip. In particular, adhesive properties derived from AFM using modified tips can be used to study specific interactions between molecules on the probe tip and the sample surface.2,3 Such modified tips can also be applied to other AFM techniques including lateral force microscopy, phase imaging microscopy, and force curve mapping to identify molecules on a sample surface by adhesive property analysis.2,3 For example, tips modified with proteins have been applied to measure the binding force of biological materials4-6 and tips modified with self-assembled monolayers have been used to identify chemical functional groups.7-10 Force curves obtained by AFM are force measurements carried out by monitoring the deflection of the cantilever as its tip approaches toward or retracts from the sample. * To whom correspondence should be addressed. † Present address: First R&D Division, R&D Headquarters, Nikon Corporation, 1-6-3 Nishi-ohi, Shinagawa-ku, Tokyo 140-8601, Japan. Telephone: +81-3-3773-1111 (ext. 2732). Fax: +81-3-37736376. E-mail:
[email protected]. (1) Binning, G.; Quate, C. F.; Gerber Ch. Phys. Rev. Lett. 1986, 56, 930. (2) Frisbie, C. D.; Rozsnyai, L. F.; Noy, A.; Wrighton, M. S.; Lieber, C. M. Science 1994, 265, 2071. (3) Akari, S.; Horn, D.; Keller, H.; Schrepp, W. Adv. Mater. 1995, 7, 549. (4) Florin, E.-L.; Moy, V. T.; Gaub, H. E. Science 1994, 264, 415. (5) Moy, V. T.; Florin, E.-L.; Gaub, H. E. Science 1994, 266, 257. (6) Lee, G. U.; Chrisey, L. A.; Colton, R. J. Science 1994, 266, 771. (7) van der Vegte, E. W.; Hadziioannou, G. Langmuir 1997, 13, 4357. (8) Wenzler, L. A.; Moyes, G. L.; Raikar, G. N.; Hansen, R. L.; Harris, J. M.; Beebe, T. P., Jr. Langmuir 1997, 13, 3761. (9) Ito, T.; Namba, M.; Bu¨hlmann, P.; Umezawa, Y. Langmuir 1997, 13, 4323. (10) McKendry, R.; Theoclitou, M.-E.; Rayment, T.; Abell, C. Nature 1998, 391, 566.
In addition to adhesion,11 force curves include other surface properties such as electric potential (double-layer force)12 and various mechanical properties.13 Such surface properties extracted from force curves obtained for each point in a scanned area can be mapped to determine their distributions. This is referred to as force curve mapping. Such distributions of surface properties including adhesion force,14-22 elasticity,17,19,21,23,24 topographic height,19,23,25 and electrical double-layer force26 have been obtained in various research fields. Adhesion force mapping using an AFM tip modified with a protein has also been applied to detect a protein on a polystyrene surface.27 (11) Weisenhorn, A. L.; Maivald, P.; Butt, H.-J.; Hansma, P. K. Phys. Rev. B 1992, 45, 11226. (12) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Nature 1991, 353, 239. (13) Burnham, N. A.; Colton, R. J. J. Vac. Sci. Technol. A 1989, 7, 2906. (14) Kaneko, R.; Nonaka, K.; Yasuda, K. J. Vac. Sci. Technol. A 1988, 6, 291. (15) Mizes, H. A.; Loh, K.-G.; Miller, R. J. D.; Ahuja, S. K.; Grabowski, E. F. Appl. Phys. Lett. 1991, 59, 2901. (16) Radmacher, M.; Cleveland, J. P.; Fritz, M.; Hansma, H. G.; Hansma, P. K. Biophys. J. 1994, 66, 2159. (17) Baselt, D. R.; Baldeschwieler, J. D. J. Appl. Phys. 1994, 76, 33. (18) Torii, A.; Sasaki, M.; Hane, K.; Okuma, S. Sens. Actuators A 1994, 44, 153. (19) Radmacher, M.; Fritz, M.; Cleveland, J. P.; Walters, D. A.; Hansma, P. K. Langmuir 1994, 10, 3809. (20) van der Werf, K. O.; Putman, C. A. J.; de Grooth, B. G.; Greve, J. Appl. Phys. Lett. 1994, 65, 1195. (21) Koleske, D. D.; Lee, G. U.; Gans, B. I.; Lee, K. P.; DiLella, D. P.; Wahl, K. J.; Barger, W. R.; Whitman, L. J.; Colton, R. J. Rev. Sci. Instrum. 1995, 66, 4566. (22) Cappella, B.; Baschieri, P.; Frediani, C.; Miccoli, P.; Ascoli, C. Nanotechnology 1997, 8, 82. (23) Radmacher, M.; Fritz, M.; Kacher, C. M.; Cleveland, J. P.; Hansma, P. K. Biophys. J. 1996, 70, 556. (24) Laney, D. E.; Garcia, R. A.; Parsons, S. M.; Hansma, H. G. Biophys J. 1997, 72, 806. (25) Mate, C. M.; Lorenz, M. R.; Novotny, V. J. J. Chem. Phys. 1989, 90, 7550. (26) Miyatani, T.; Horii, M.; Rosa, A.; Fujihira, M. Appl. Phys. Lett. 1997, 71, 2632. (27) Chen, X.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Langmuir 1997, 13, 4106.
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Although much information can be derived by force curve mapping, it requires the analysis of a huge amount of force curve data. Analyzing these data and extracting surface properties from it are complicated and timeconsuming. To overcome this, we have developed an automatic data-processing algorithm to analyze force curves and extract surface properties.28 Chromosomes are threadlike bodies carrying genetic information and are known to have a hierarchical structure. Chromosome structure has been studied using several methods including X-ray diffraction analysis, electron microscopy,29-31 and atomic force microscopy.32,33 In this paper, we have applied force curve mapping to observe the chromosome’s complex structures. We report on artifacts that appeared in adhesion force images obtained in our observations and their origin. A similar artifact in the force curve measurement was reported by Hoh et al.34 We also discuss briefly possible solutions to avoid such artifacts. II. Materials and Methods We employed a custom-built AFM with three stacked piezo actuators.35 Our AFM was controlled by a system consisting of a digital signal processor and a computer. After an interesting location was identified by optical microscopy, an entire set of force curve data for mapping was acquired at that location with the AFM. Each data set included 128 × 128 force curves and was stored by the computer. Through data processing with our own software,28 the force curves obtained were analyzed, and adhesion forces, topographic heights, and elasticities were derived. These surface properties were then mapped. The force curves were obtained by vertically moving a cantilever at a speed of about 50 µm/s over a maximum travel height of 1.5 µm. The force curve acquisition rate was about 10 Hz. When the cantilever deflection exceeded a given load of 10 nN during approach, the control software automatically began retracting the cantilever. The silicon nitride cantilevers used had a force constant of 0.1 N/m and a tip radius of 50 nm (Microlever; Park Scientific Instrument, Inc.). The experiments were conducted in air. A human chromosome sample was prepared by a surfacespreading whole-mount technique.29,30 Cultured human lymphocytes were spun down and placed on a water surface where the cells burst. Consequently, the chromosomes were released from the cells and spread as a thin film on the water surface. This thin film was transferred to a silicon substrate where it dried and was used as a sample.
III. Results Figure 1 shows three topographic images on the left paired with their respective adhesion force images on the right obtained for the same chromosome sample. Each pair of images was derived from one set of force curve data; that is, those images were obtained simultaneously. Two chromosomes were observed in the square scan area of 25 µm shown in parts a and b of Figure 1. Fibrous structures were seen on the substrate around these chromosomes. Parts c and d of Figure 1 were obtained by zooming into the white square indicated in Figure 1a. The fibrous structures were more clearly recognized in Figure 1c. Further zoomed topographic and adhesion force (28) Sugisaki, K.; Nakano, K.; Sugimura, H.; Kandaka, N.; Nakagiri, N. Jpn. J. Appl. Phys. 1998, 37, 3820. (29) Gall, J. Science 1963, 139, 120. (30) Watanabe, M.; Tanaka, N. Jpn. J. Genet. 1972, 47, 1. (31) Hozier, J.; Renz, M.; Nehls, P. Chromosoma (Berl.) 1977, 62, 301. (32) De Grooth, B. G.; Putman, C. A. J. J. Microscopy 1992, 168, 239. (33) Kinjo, Y.; Shigeno, M.; Shinohara, K.; Watanabe, M. Cytologia 1996, 61, 327. (34) Hoh, J. H.; Engel, A. Langmuir 1993, 9, 3310. (35) Nakano, K. Rev. Sci. Instrum. 1998, 69, 1406.
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images are shown in parts e and f of Figure 1, respectively. The topographic image indicates that each fibrous structure consists of many round structures about 100 nm in diameter. We believe that these round structures are nucleoprotein beads (i.e., superbeads31) which are known to be several tens of nanometers in diameter and have previously been observed by several methods. The bead size obtained from our observation here is different from that reported. It is possible that the resolution of our bead images was limited by the AFM tip diameter (100 nm). In the adhesion force image, less adhesive regions, which are seen as dark structures, are observed near locations corresponding to beads in the topographic image. The adhesion force between the tip and the sample depends on their respective materials and curvatures. As the curvature of one or both of the structures increases, the adhesion force decreases. Thus, the dark, less adhesive regions correspond to the tops of the beads observed in the topographic image. However, the locations of these less adhesive regions are slightly different from those of the corresponding beadssthere is an overall downward shift to varying degrees. Figure 2 shows cross sections from the topographic image taken across one bead location and its corresponding less adhesive region, on the adhesion force image as indicated respectively in parts e and f of Figure 1 by A-A′. The convex structure in Figure 2a indicated by the arrow represents the bead, and the concave structure in Figure 2b indicated by the arrow shows the less adhesive region corresponding to that bead. The position of lowest adhesion force measured in this cross section (i.e., at the bottom of the concave structure) is shifted by about 80 nm from that of the highest point on the corresponding topographic cross section. In addition, this less adhesive region has an asymmetric structure with different slopes on each side. The cantilever was positioned with its tip to the left and its base to the right relative to these cross sections. The location shift seen in the adhesion image occurred toward the left, that is, in the direction away from the cantilever base. IV. Discussion In our experiments, the locations of the less adhesive regions shifted from the corresponding regions on the topographic images. Assuming that the friction between the tip and the sample surface was negligibly small, this phenomenon can be explained by lateral tip movement due to cantilever bending. Figure 3 shows schematic drawings of the cantilever in various states during which adhesion force and topographic height are measured. During force curve measurements, the cantilever base at position C on the y axis moves vertically on the z axis. The cantilever approaches the sample surface, and its tip makes contact. Topographic height is measured as the z position of the cantilever base, when there is no cantilever deflection and the tip is in contact with the sample at position A on the y axis (Figure 3a). When the cantilever is retracted from the surface, the cantilever bends downward because of the adhesion force between the tip and the sample. Due to this downward deflection, the tip slides toward the cantilever base in the positive direction on the y axis shown in Figure 3b. When the retracting force eventually exceeds the adhesion force, the tip detaches at position B on the sample (Figure 3c). Since adhesion force is derived from the cantilever deflection just before tip detachment, adhesion force is measured at position B, in contrast to the topographic height measured at position A. Since the tip slides as the cantilever bends,
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Figure 1. Topographic and adhesion force images obtained of chromosomes on a Si substrate. (a) Topographic and (b) adhesion force distribution images derived from a 25-µm square scanned area. c and d show the same, respectively, for the zoomed area of 5.8 µm indicated by the white square in a. e and f show further zoomed images of about 1.4 µm indicated by the white square in c.
the resulting shift in location between the topographic and adhesion measurements increases as the cantilever bending increases. The shift distances between the positions of the tops of the beads and those of minimum adhesion in the corresponding less adhesive regions were measured from parts e and f of Figure 1 and plotted as a function of the cantilever deflections just before detachment. Cantilever deflection was derived from adhesion, that is, the adhesion force divided by the cantilever spring constant. The resulting
plot shown in Figure 4 shows a correlation between the shift distance and cantilever deflection. To analyze the artifacts appearing in the adhesion force images, we considered artifacts appearing for the simple flat surface with different adhesive regions (Figure 5). In our force curve mapping, the tip is moved to on an appropriate position above the sample where a force curve is to be measured. The force curve measurement begins at position P1 on the y axis in Figure 5, where the tip first contacts the sample before cantilever deflection begins.
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Figure 4. Distribution of shift distance as a function of cantilever deflection at the point of lowest adhesion force measured locally. The solid line indicates the geometrically estimated shift distance due to cantilever deflection.
Figure 2. Cross sections of (a) topography and (b) adhesion force obtained for A-A′ indicated in parts e and f of Figure 1.
Figure 5. Schematic diagrams of the relationship between (a) the assumed adhesive structure and (b) the resulting adhesive structure which would be measured. Figure 3. Schematic drawings of the three states of the cantilever. (a) The tip is in contact with the sample without deflection of the cantilever. (b) The cantilever bends downward due to adhesion force. (c) The cantilever retracting force exceeds the adhesion force, and the tip detaches from the sample.
The topographic height derived from this force curve measurement is for location P1. When the cantilever retracts from the sample, it starts bending downward and its tip moves toward its base. The line P1-Ap in Figure 5a indicates the cantilever tip sliding toward the cantilever base as the adhesive force between the tip and the sample increases. The retracting force finally exceeds the adhesion force at position P2 where the tip detaches from the sample. The adhesion force derived from this force measurement is for this position P2. Thus, this force curve gives height information at P1 and adhesion force at P2. In the same way, when the force curve is measured at position Q1, the height at Q1 is derived. As the cantilever retracts, the tip slides as indicated in Figure 5a by the
line Q1-AQ. The tip cannot detach at the small, highly adhesive region indicated by AHigh because of its high adhesion. After the tip crosses the highly adhesive region, it detaches from the surface at Q2. Therefore, this force curve gives the adhesion force AQ at Q2, which is not the actual point of the highest adhesion AHigh. In other words, the information of the highest adhesive property is missing as indicated by the dotted lines shown in Figure 5b. Similarly, the force curve measured at position R1 gives height information at R1 and adhesion force at R2. Since the tip easily detaches from the low adhesive region, the lowest adhesion force ALow can be measured. As a result, when the adhesion forces are mapped for the locations at which the force curve measurements were taken, the position of the measured adhesion force structure is shifted, and its shape is asymmetrically deformed. In addition, lower adhesive properties can be easily detected but higher adhesive properties may be lost. The shift distance can be geometrically estimated. The solid line in Figure 4 shows the results obtained when the
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shift distance is estimated using our experimental conditions (the cantilever length ) 140 µm and the lever attatchment angle ) 10°). According to this estimation, the shift distance varied from 30 to 70 nm for cantilever deflection between 200 and 400 nm. The actual shift distances obtained from our experiment ranged from 10 to 110 nm for cantilever deflection between 200 and 400 nm. Since the estimated results qualitatively agree with our experimental data, we conclude that cantilever deflection was the major reason for the artifacts observed. A similar artifact caused by the cantilever deflection in a force curve measurement was reported by Hoh et al.35 As mentioned by them, several methods could be employed to eliminate these artifacts. Since these artifacts are originally due to cantilever bending or tip sliding, we can avoid these artifacts altogether by preventing the cantilever from bending or the tip from sliding. Direct force measurement using electrostatic36 or magnetic force37 could be employed to prevent the cantilever from bending. Using the force sensor which is designed symmetrically (36) Joyce, S. A.; Houston, J. E. Rev. Sci. Instrum. 1991, 62, 710. (37) Stewart, A. M.; Parker, J. L. Rev. Sci. Instrum. 1992, 63, 5626.
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for the tip along the z axis or compensating the tip lateral motion by the AFM control system could be also employed. V. Conclusions We observed artifacts in adhesion force images obtained by force curve mapping. Through qualitative simulation, tip sliding due to cantilever deflection was determined to be the major reason for these artifacts. These artifacts not only make it difficult to identify corresponding positions between the topographic and adhesion force images obtained but also increase the risk that small, highly adhesive regions might be missed. This is a potentially serious problem for molecular-size applications including molecular recognition by detecting high adhesion force due to a specific tip/sample interaction. These artifacts may be eliminated by applying methods that either prevent cantilever deflection, employ a symmetrical force sensor, or adjust cantilever position to compensate for tip movement. Acknowledgment. We thank Takuma Yamamoto and Hisao Fujisaki for insightful discussions. LA980746Z
