Langmuir 1995,11, 1711-1714
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Artifacts in Atomic Force Microscopy Images of Fine Particle and Protein Two-Dimensional Crystals As Evaluated with Scanning Electron Microscopy and Simulations Tetsuya Miwa, Mariko Yamaki, Hideyuki Yoshimura, Satoshi Ebina, and Kuniaki Nagayama* Nagayama Protein Array Project, ERATO, JRDC, 5-9-1 Tokodai, Tsukuba, Ibaraki 300-26, Japan Received January 3, 1994. In Final Form: January 27, 1 9 9 9 With the aid of simulations and a scanning electron microscope equipped with a cold field emission source (FE-SEM),we evaluated artifacts in atomic force microscopy (AFM)images by using two-dimensional (2D) crystals of four types of particles that ranged in size from 12 to 144 nm. The AFM images of these 2D crystals were taken in air using an atomic force microscope equipped with a grown carbon tip whose apex was 15-20 nm in diameter. The SEM images obtained for the same 2D crystals indicated discernible spheres that were hexagonally arranged for all crystals. We took these SEM images as references for evaluating the AFM images. The AFM images for the 2D crystals of the 144 nm particles were identical to the SEM images. However, in the AFM images, we identified artifacts that gradually increased as the particle size decreased. Thus, we found that one cause of the artifacts that often occur in fine topography is the use of a probe whose tip diameter is comparable in size to the topography of the objects. In our simulations of the AFM imaging, we assumed four types of hexagonal lattices of particles and a spherical probe tip of 20 nm diameter. Our simulations reproduced the same phenomenon seen in the actual AFM images, namely, the increase in artifacts as the particle size decreases. Nevertheless, in these simulations, the image of the 2D crystals of 12 nm particles was still identified. Furthermore, in the same simulations, when random oscillationsof either the tip or the particles were assumed to occur during the scanning over a range of a few nanometers, the simulated images were very similar to the real AFM images. Based on our results, we concluded that the artifacts identified in the real AFM images are produced by such random displacements (or oscillations) and by the effect of the finite size of the probe tip.
1. Introduction Many sophisticated functions of structures such as biological structures are tightly related to their topography a t the nanometer scale. Therefore, atomic force microscopy (AFM) imaging has been extensively applied to imaging the topography of these structures.lg2 An intolerable problem is that artifacts appear in the AFM images as the topography of the objects approaches the size of the probe tip used in the imaging.3 Consequently, it is recommended to estimate artifacts in AFM images by imaging known structures first, and then imaging the unknown structure^.^-^ Wanget a1.’ and Li and Lindsay reported that AFM imaging (taken in air) of spin-coated films of 100 nm diameter polystyrene particles provides clear images of the particles by using a probe tip whose diameter is about 50 nm. The AFM imaging of known biological structures, such as globular proteinsgand cyclic DNAs,lO demonstrates that the resolution ofAFM imaging noticeably deteriorates due to physical contact of the probe tip with object surfaces. Although this deterioration Abstract published in Advance A C S Abstracts, April 1, 1995. (1) Hansma, P.K.; Elings, V. B.; Marti, 0.;Bracker, C. E. Science 1988,242,209. (2)Zenhausern, F.; Adrian, M.; Emch, R.; Taborelli, J.; Jobin, M.; Descouts. P.Ultramicroscow 1992.42-44. 1168. (3)Miwa, T.; Yamaki, M::Yoshimura, H.’; Ebina, S.; Nagayama, K. Jpn. J . Appl. Phys. 1992,31,1495. (4) Keller, D. Surf. Sci. 1991,253,353. (5)Radmacher, M.;Tillmann, R. W.; Fritz, M.; Gaub, H. E. Science 1992,257,1900. (6)Keller, D. J.; Franke, F. S. Surf. Sci. 1993,294,409. (7)Wang, Y.;Juhu6, D.; Winnik, M. A,; Leung, 0. M.; Goh, M. C. Langmuir 1992,8, 760. (8) Li, Y.; Lindsay, S. M. Reu. Sci. Instrum. 1991,62,2630. (9)Eppell, S. J.; Zypman, F. R.; Marchant, R. E. Langmuir 1993,9, 2281. (10) Bustamante, C.;Vesenka, J.;Tang, C. L.; Ress, W.; Guthold, M.; Keller, R.Biochemistry 1992,31,22. @
suggests that this physical contact induces changes in many properties, such as surface topography5 and surface charges of the objects and of the probe tips, no report provides clues on how to eliminate artifacts that accompany AFM imaging. To evaluate such artifacts when imaging fine topography, however, there are two requirements. First, real images or “pseudoreal” images must be made so that comparisons can be made to a reference structure. Second, the structures to be imaged should have regular, fine topography ranging from 10 to 100 nm (which is the scale of biopolymers) so that the structures can be distinguished from the bare surfaces. In this study, we evaluated artifacts in AFM images for topography in which the size range of the objects was on the same order as the size of the probe tip. For reference imaging, we used scanning electron microscopy (SEM) with a microscope that was equipped with a field emission source (FE-SEMI because it provided real images of fine topography, such as that seen in biological structures.l’ For the AFM imaging, we used a n AFM probe that was equipped with a grown carbon tip, with a diameter of 1520 nm. As our reference samples, we used two-dimensional (2D) crystals of four types of fine particles: polystyrene (PSt)particles (144, 55, and 38 nm in diameter) and holoferritin12 (an iron-storage protein, 12 nm in diameter). To gain further insights into the mechanism of artifacts in AFM, we also did simulations of our AFM imaging experiments. 2. Experimental Section The PSt particles with diameters of 144 & 2 and 55 & 3.6 nm were purchased from JSR Co., and those with a diameter of 38 =k 13.9 nm were from Interfacial Dynamics Co. Horse spleen (11)Nagatani, T.; Saito, S.;Sato, M.; Yamada, M. Scanning Microsc. 1987,1, 901.
0743-746319512411-1711$09.00/0 0 1995 American Chemical Society
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Figure 1. SEM and AFM images of 2D crystals of polystyrene particles on glass and ferritin molecules on mica. The upper row shows the SEM images of 144f2,55 f3.6, and 38 f 13.9nm PSt particles (a,b, and c, respectively) and the 12nm ferritin molecules (d). The lower row shows the corresponding AFM images and diffraction spots of the same crystals, respectively. holoferritin was purchased from Boehringer Mannheim. The 2D crystalsof these PSt particles were produced directly on glass surfacesby the convective assemblymethod13.14and imaged using an atomic force microscope and a scanning electron micrscope. For the AFM and SEM imaging, a portion of these crystals was then coated with carbon by using a vacuum evaporator (JEE4X, JEOL). The 2D crystals of ferritin were produced directly on clean surfaces of mercury in pure oxygen gas as previously reported.15 For one series of AFM and SEM imaging, a portion of the 2D crystals of ferritin was then transferred to a surface of freshly cleaved, carbon-coated mica. For another series, to obtain homogeneous surfaces of the 2D crystals for the AFM imaging, we reinforced the crystals by covering them with a polymerized methane-ethylene membrane, about 1nm thick, using a plasma polymerizer (PNR-110, Ushio, Inc.).16 The atomic force microscope we used was an SFA-300 model (Seiko Instruments Inc.) equipped with a piezoelectric xyztranslator with a scanning span of 20 x 20 x 2 pm (with a single step of 0.4 x 0.4 x 0.05 nm). For the AFM imaging, we used a carbon probe tip4J7J8that was newly grown on the apex of a commercial AFM cantilever made of Si3N4 (Microlever, Park Scientific Instruments) using a scanning electron microscope (JSM-820,JEOL).lg The probe tip had a cone-shapedapex with a diameter of 15-20 nm, as determined using SEM.lg The imagingwas carried out under a constant forcemode in ambient air at a controlled relative humidity of 30-60%. The bending force of the cantilever was minimized by adjusting the distance between the tip and the object surface. This forcewas calculated (12)Banyard, S.H.; Stammers,D. IC;Harrison, P. M. Nature 1978, 271,282. (13)Dushkin, C. D.; Nagayama, K.; Miwa, T.; Kralchevsky, P. A. Lungmuir 1993,9,3695. (14)Velev, 0.D.; Denkov, N. D.; Paunov, V. N.; Kralchevsky, P. A.; Nagayama, K.hngmuir 1993,9,3702. (15)Yoshimura, H.; Matsumoto, M.; Endo, S.; Nagayama, K. Ultramicroscopy 1990,32,265. (16)Tanaka,A.;Yamaguchi,Y.;Iwasaki,T.; Iriyama,K. Chem.Lett. (Jpn.) 1989,1219. (17) Lee, K. L.; Hatzakis, M. J . Vac. Sci. Technol. 1989,B7, 1941. (18)Akama, Y.; Nishimura, E.; Sakai, A.; Murakami,H. J . Vac.Sci. Technol. 1990,A8, 429. (19)Yamaki, M.; Miwa, T.; Yoshimura, H.; Nagayama, K. J . Vac. Sci. Technol. 1992,BlO, 2447.
from the spring constant of the cantilever, greater than N. The data of the AFM images were converted to Tag Image File Format, transferred to a UNM workstation, and then transformed by Fourier analysis using EMIDO image processing software (developed in-house). The resulting patterns of the diffraction spots (calculated from the Fourier transforms) were useful in evaluating the type and spacing of the lattices of the particles in the images. The field emission scanning electron microscope we used was an S-5000H(Hitachi)operated at an acceleration voltage of 1.01.2 kVand an emissioncurrentof 1OpA. Under these conditions, optimal resolution at 1kV was guaranteed to 2.5 nm. Using a recursive filter, we integrated 64 SEM images at a TV-scan rate and recorded them on Polaroid film. We then digitized some of the images using either a CCD camera (512 x 512 pixels) or an image scanner and then transformed them by Fourier analysis using EMIDO. To study the mechanism of the artifacts present in the AFM images of the 2D crystals, we did simulations of these images. In these simulations, we assumed both the model particles and the probe tip to be spherical. We then set up a hexagonal lattice (on an x-y plane) with lattice spacing of 144,55,38, or 12 nm, and centered the model particles of 144, 55, 38, or 12 nm, respectively, on these lattice points. We set the diameter of the probe tip at 20 nm. We calculated the z-coordinate of the probe tip during the scanning of the surface of the model particles using the equation, z = r1 + r2 - ((rl+ r2I2 - d 2 P ,where rl is the radius of a particle on the lattice, r2 is the radius of the probe tip, and d is the distance from the nearest particle center to the probe tip on thex-y plane. We note that our simulations modeled the scanning of topography in which the size of the objects was equivalent to that of the probe tip; therefore, we did not account for the cone effect that occurs when the side of the probe tip comes in contact with the object^.^ In another series of simulations using the same conditions, we included random displacements of particles (and/or tip position) by giving random oscillations of 1-10 nm to the particle centers.
3. Results
SEM Images. Figure la-d showsthe SEM images of the 2D crystals for the 144,55, and 38 nm PSt particles
Evaluation of Artifacts in AFM Images
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Figure 2. Simulationresults of the AFM imagesfor 2D crystals of 144,55,38,and 12nm spheres for a probe tip 20 nm in diameter. The centers of these model spheres were placed on the lattice points that were arranged on an x-y plane which had hexagonal lattices with lattice spaces of 144,55,38,and 12 nm. The upper row shows the images of the 144,55,38,and 12 nm spheres (a, b, c, and d, respectively) when no random oscillations of the probe tip were included in the simulations. The indicators of the depth are indicated along the z-axis (in nanometers). The lower row shows the simulated images and corresponding diffraction spots for the same spheres (e, f, g, and h, respectively) when 3 nm random oscillations of the tip were included in the simulations. on glass and the 12 nm ferritin molecules on mica, irregularly arranged (Figure lh), and again, no diffraction respectively. Although it was sometimes difficult to obtain spots were evident. We note that this AFM image of clear images of the particles that were not carbon-coated, ferritin 2D crystals was different from that of the both the PSt particles and the ferritin molecules were corresponding SEM image (Figure Id). identified to be spheres that were hexagonally arranged. Simulations of the AFM Imaging. The upper row (We found no discernible effects of the carbon coating on of Figure 2 shows the simulated AFM images of the four the images we obtained.) The hexagonal spacingwas more types of particles. The contour, or depth, shading is seen clearly demonstrated in the diffraction spots as seen in along the z-axis. For all crystals examined, these simulaFigure 1. We note here that SEM images of the bare tions clearly indicated spheres that were hexagonally surfaces of both the glass and the mice indicated no arranged. Nevertheless, the resolution along the z-axis patterns of regular structures, showing that the patterns deteriorated as the size of the spheres decreased, a observed in the images of the particles were due only to condition also seen in the difiaction spots. In the the particles or molecules themselves. simulations that assumed a probe tip of 50 nm diameter, AFM Images. The AFM imagingwas done on the same we obtained the same hexagonal images, although the samples as in the SEM images, however, with and without resolution slightly deteriorated along the z-axis. Therethe carbon coating. There was no difference in the images fore, with the growing carbon tip that we used in our real due to the carbon coating. Figure le-g shows the AFM AFM imaging in this study (the diameter of the apex was images of the particles without coating and their respective 15-20 nm), we should have obtained images that were computed diffraction spots. Figure l h shows the image close to the simulatedAFM images (Figure lh). However, of the ferritin crystals. The AFM image of the 144 nm this was not the case. PSt particles indicated spheres that were hexagonally In contrast, using the simulationsthat included random arranged with 144 nm lattice spacing (Figure le). This oscillations (ranging from 1 to 10 nm) of the sphere regularity of the lattice was more clearly identifiedin the positions from the lattice points, we reproduced images diffraction spots (the inset in Figure le). The image of that were similar to the real AFM images. Moreover, we , the 55 nm PSt particles also indicated discernible hexobtained the same results when these oscillations were agonal lattice of the spheres (Figure 10. Nevertheless, given to the probe tip rather than to the particles. The this image is slightly deteriorated compared with that of lower row of Figure 2 shows the AFM images from the the 144 nm particles. This deterioration can also be simulations that were made with 3 nm random oscillaidentified in the diffraction spots (see the inset). For the tions. Particularly in the image of the 12 nm particles 144 and 55 nm PSt particles, AFM provided images that (Figure 2h), the 3 nm displacements produced remarkable were similar to the SEM images. In contrast, the AFM deterioration in the image, as seen in the disappearance images of the 38 and 12 nm PSt particles contained of the hexagonal lattice. We emphasize that only the significant artifacts (Figure lc,d, respectively). The 38 simulations that included random displacements (for nm PSt particles were not clearly identified by AFM; either the probe tip or the particles) of a few nanometers furthermore, the diffraction spots in this AFM image reproduced images that were similar to the real AFM indicated no hexagonal arrangement. The AFM image of ferritin showed particles 10-20 nm in diameter that were images.
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4. Discussion The SEM images indicated discernible hexagonal lattices for all of the 2D crystals of the particles that we examined in this study (Figure la-d). Because of the resolution, we focused our attention only on the morphology of spheres, namely, the lattice type and the lattice spacing. As seen in Figure 1, the lattice type and spacing in these SEM images were clearly obtained. The comparison of the AFM images (the upper row of Figure 1) with the SEM images (the lower row of Figure 1)clarified that the AFM images of the 2D arrays of particles with diameters greater than 50 nm are close to the real images, while those of smaller particles (less than 38 nm) contain discernible artifacts. Can these artifacts in AFM images be attributed to the interactions of the carbon tip with different types of materials present in the same scanning, such as the protein and the carbon-coated surfaces, as was reported for a silicon nitride tip interacting with protein on mica?g To examine this possibility, we imaged the ferritin 2D crystals that were homogeneously covered with a plasmapolymerized membrane (as described in the Experimental Section). As a result, however, we identified no change in the AFM images obtained. Therefore, we can conclude that the artifacts observed in AFM images are due mainly to physical contact of the probe tip with the object surface. Our simulations revealed causes of the artifacts. Without the random oscillations, these simulations clearly yielded hexagonal images for all model crystals examined (the upper row of Figure 2). At the same time, however, we identified increasing deterioration in the resolution along the z-axis corresponding to a decrease in the sphere
size (Figure 2a-d). This indicates that one cause of the artifacts is the use of a probe tip whose size is comparable to the size of the topography. Although the use of a thicker probe tip (Le., 50 nm in diameter) further deteriorated the images, we still obtained hexagonal images for all crystals (Figure 2a-d). Therefore, the mechanism of the artifacts involves more than one factor. Another clue was revealed by our simulations that accounted for the random oscillations (a few nanometers) of the probe tip or particles that occur during the scanning (lower row of Figure 2). These simulated images were very similar to the real images. Therefore, we strongly suggest that a random displacement of either the probe tip or the surface of the objects also creates the artifacts seen in the real AFM images. Because reinforcing the ferritin 2D crystals with a polymerized membrane did not improve the real image, we further conclude that the random displacements are mainly attributed to the probe tip and not to physical distortion in the surfaces of the objects. From our results, we claim that artifacts seen in AFM imaging are caused by random displacements of the probe tip during scanning, in conjunction with the effects that occur when the size of the probe tip approaches that of the topography of the objects.
Acknowledgment. The plasma polymerization was kindly made by Dr. A. Tanaka a t Nippon Laser and Electronics Lab. Dr. R. Lewis (Tsukuba Research Consortium, Tsukuba, Japan) has kindly critiqued this manuscript. LA940020C
