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An Atomic Force Microscopy Study for the Assembly Structures of Tobacco Mosaic Virus and Their Size Evaluation Hideatsu Maeda JRCAT/National Institute for Advanced Interdisciplinary Research and National Institute of Bioscience and Human-Technology, 1-1-4 Higashi, Tsukuba, Ibaraki 305, Japan Received December 16, 1996. In Final Form: April 18, 1997X Assemblies of tobacco mosaic virus (TMV) dried on glass substrates showed characteristic patterns which were found to be related to (1) the concentration, (2) the anisodimensional shape, and (3) the semiflexibility of the virus. The arrangements of the virus particles in the patterns were investigated using an atomic force microscope (AFM). In the dried assemblies of TMV at a concentration of 50 mg/mL, characteristic birefringent crack patterns were observed, including highly oriented regions in which the particles were uniaxially oriented but longitudinally at random, and zigzag pattern regions in which sharply bent particles having a critical bending angle of 60° were found, indicating that the individual TMV particles are semiflexible rods rather than hard rods. In these regions, the interparticle end-to-end connections were frequently observed. In addition, several types of defects were observed in the crack patterns. In the dried assemblies for a TMV concentration of 0.5 mg/mL, characteristic 2D-network patterns were observed, which were formed by branching and fusing of the 2D-spindle tactoids of TMV, as well as a periodic pattern which may have resulted from spatially nonuniform speeds of the peripheral boundary of the suspension droplets during drying. The particle arrangements of the branched and fused spindles were also observed. The size of the TMV particle in the patterns was measured using the tapping mode imaging of the AFM: the length and width of the particles in the highly oriented regions were 301 and 14.7 nm, respectively, and the heights of the particles in the 2D-networks were 16.8-18.6 nm, indicating that the particles are not crushed. These values agree well with earlier ones estimated by X-ray diffraction and electron microscopy measurements. This shows that the TMV particles, in particular their highly oriented assemblies, can be used as standard specimens for the tapping mode imaging of the AFM in the scale of 10-100 nm. It was also demonstrated that single TMV particles fixed on substrates can be used to evaluate the radii of the tip apexes.
Introduction Applications of atomic force microscopy (AFM) to biological materials (such as proteins, biomembranes, and DNA) have increased remarkably in recent years.1 It has been shown that AFM has an ability to obtain nanoscale topographic images even for biomolecules in various environments (e.g., air, vacuum, and solvents) without any complicated sample pretreatment. However, the interactions between the AFM probe tips and isolated molecules on the substrates frequently induce displacements and deformations of the molecules. In addition, the shape and volume of the probe tip result in an artificial broadening of the true topographic images of the molecules. We thought that a high ordering of the molecules must be effective in avoiding these effects, because in ordered structures each molecule is tightly fixed to one another and the tip apex alone can interact with the surface of the molecules. Thus, to prevent the inclusion of such artifacts in the AFM images, we tried to highly order the tobacco mosaic virus (TMV) particles, particularly by using the concentrated suspensions on glass substrates. So far, using AFM in the contact mode, the dimensions of the TMV particles from dilute suspensions have been measured on various kinds of substrates (e.g., mica,2-6 X
Abstract published in Advance ACS Abstracts, June 15, 1997.
(1) Linsay, S. M. In Scanning Tunneling Microscope and Spectroscopy; Donnell, D. A., Ed.; VHC: New York, 1992, p 335. (2) Zenhausern, F.; Adrian, M.; Emch, R.; Taborelli, M.; Jobin, M.; Descouts, P. Ultramicroscopy 1992, 42, 1168. (3) Thundat, T.; Zheng, X.-Y.; Sharp, S. L.; Allison, D. P.; Warmash, R. J.; Joy, D. C.; Ferrrell, T. L. Scanning Microsc. 1992, 6, 9. (4) Zenhausern, F.; Adrian, M.; ten Heggeler-Border, B.; Ardizzorni, F.; Descouts, P. J. Appl. Phys. 1993, 73, 7232. (5) Wadu-Mesthrige, K.; Pati, B.; Martin McClain, W.; Lui, G.-Y. Langmuir 1996, 12, 3511.
S0743-7463(96)02105-1 CCC: $14.00
silicon wafer,7 polylysine-coated glass,8 albumin-coated mica,5 and a mixed hydrocarbon/fluorocarbon LangmuirBlodgett (LB) film9,10), in different environments (air, 2-propanol,8 and 2-butanol5), and with several types of probe tips (pyramidal, conical, and electron beam deposited tips).4 TMV is a rodlike virus with a length of 300 nm.11-16 The virus is composed of structurally equivalent protein units set in a helical array around the long axis of the particle.17,18 The pitch of the helix (on which the protein subunits lie) is 2.3 nm.14,16,19-21 The external surface of the particle has grooves about 3 nm deep between succesive helical turns.22,23 The maximum diameter of the particle is 18 nm,14,16,22-26 and the average diameter (that can be (6) Bushell, G. R.; Watson, G. S.; Holt, S. A.; Myhra, S. J. Microsc. 1995, 180, 174. (7) Imai, K.; Yoshimura, K.; Tomitori, M.; Nishikawa, O.; Kokawa, R.; Yamamoto, M.; Kobayashi, M.; Ikai, A. Jpn. J. Appl. Phys. 1993, 32, 2962. (8) Karrasch, S.; Dolder, M.; Scharbert, F.; Ramsden, J.; Engel, A. Biophys. J. 1993, 65, 2437. (9) Frommer, J.; Luthj, R.; Meyer, E.; Anselmetti, D.; Dreler, M.; Overney, R.; Guntherodt, H.-J.; Fujihira, M. Nature 1993, 364, 198. (10) Anselmetti, D.; Dreier, M.; Luthi, R.; Richmond, T.; Mayer, E.; Frommer, J.; Gruntherodt, H.-J. J. Vac. Sci. Technol. 1994, B12 (3), 1500. (11) Stanley; W. M.; Anderson, T. F. J. Biol. Chem. 1941, 139, 325. (12) Williams, R. C.; Steere, R. C. J. Amer. Chem. Soc. 1951, 73, 2057. (13) Hall, J. Amer. Chem. Soc. 1958, 80, 2556. (14) Finch, J. T. J. Mol. Biol. 1964, 8, 872. (15) Markham, R.; Hitchborn, J. H.; Hills, G. J.; Frey, S. Virology 1964, 22, 342. (16) William, R. C.; Fisher, H. W. J. Mol. Biol. 1970, 52, 121. (17) Klug, A.; Casper, D. L. D. Adv. Virus Res. 1960, 7, 225. (18) Casper, D. L. D. Adv. Protein Chem. 1963, 8, 37. (19) Watson, J. D. Biochim. Biophys. Acta 1954, 13, 10. (20) Franklin, R. E. Nature 1955, 175, 379. (21) Finch, J. T. Virology 1969, 38, 182. (22) Franklin, R. E.; Klug, A. Biochem. Biophys. Acta 1956, 19, 403.
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measured only for close-packed particles, which were shown to be intermeshed with one another due to the grooved structure of the surface) is 15 nm.22,27,28 Bernal et al.27 found that a drop of TMV suspension dried between two glass plates forms only two distinct phases: a disordered phase which is an isotropic sol and an ordered phase which varies from an anisotropic sol (through various stages of viscosity and rigidity) to a wet and finally to a dry gel. Furthermore, from X-ray analysis they determined an interparticle separation of 15.2-30 nm in the ordered phase. Recently, further studies concerning the ordered phases (nematic, smectic, and colloidal crystalline) appearing in TMV suspensions have been performed extensively.29-32 Zasadzinski et al.32 used freeze fracture electron microscopy to visualize the particle ordering of TMV suspensions. Similarly, a rodlike colloidal particle of β-FeOOH also formed a smectic structure in the highly concentrated suspensions.33-35 Onsager theoretically showed that an ordered phase can occur in rodlike particle systems with only hard core interparticle interactions.36,37 In this study, mono- and multilayered assemblies formed by drying droplets of concentrated TMV suspensions on glass and mica substrates were examined by AFM to determine: (1) What characteristic structures appear in the assemblies? Do they depend on the TMV concentration? What arrangements do the particles take in the assemblies? (2) Can we exactly determine the size of the individual particle in the assemblies by AFM? Experimental Section Tobacco mosaic virus particles, which were prepared at a concentration of 50 mg/mL in distilled water, were kindly supplied by Drs. C. Masuda and M. Suzuki. The TMV concentrations of the suspensions used were 0.5, 5, and 50 mg/mL. Droplets (20-30 µL) of the suspensions were dried on glass and mica substrates. The time required for complete drying was 20-40 min. This was controlled using glass or plastic covers over the droplets. On both substrates, the assembly patterns of the particles were slightly different macroscopically, particularly at low concentrations (around 0.5 mg/mL), but the particle arrangements were almost the same. Only the data obtained using the glass substrates are shown below, since they are more easily observed using an optical microscope. The dried samples were mounted on the piezoelectric scanner of the atomic force microscope (Nanoscope IIIa, Digital Instruments, Inc., Santa Barbara, CA). We used microfabricated cantilevers of Si3N4 (Nanoprobes, Digital Instruments, Inc.) with a total length of 123 µm and a spring constant of 27-70 N/m. Calibration of the scanner was carried out using an optical calibration grating supplied with the system used. We used two different types of tapping mode AFM imaging: “height mode” imaging, which provides height images of the (23) Namba, K.; Pallanayek, R.; Stubbs, G. J. Mol. Biol. 1989, 208, 307. (24) Casper, D. L. D. Nature 1956, 177, 475. (25) Jeng, T.-W.; Crother, R. A.; Stubbs, G.; Chui, W. J. Mol. Biol. 1989, 205, 215. (26) Smith, M. F.; Langmore, J. P. J. Mol. Biol. 1992, 226, 763. (27) Bernal, J. D.; Fankuchen, I. J. Gen. Physiol. 1941, 25, 111. (28) Matthews, R. E. F.; Horne, R. W.; Green, E. M. Nature 1956, 22, 635. (29) Fraden, S.; Casper, D. L. D.; Philips, W. Biophys. J. 1982, 37, 97a. (30) Wen, X.; Meyer, R. B.; Casper, D. L. D. Phys. Rev. Lett. 1989, 63, 2760. (31) Meyer, R. B. In Dynamics and Patterns in Complex Fluid; Onuki, A., Kawasaki, K., Eds.; Springer Proceedings in Physics; Springer Verlag: Berlin, 1990, Vol. 52, p 62. (32) Zasadzinski, J. A. N.; Sammon, M. J.; Meyer, R. B.; Cahoon, M.; Caspar, D. L. D. Mol. Cryst. Liq. Cryst. 1986, 138, 211. (33) Maeda, Y.; Hachisu, S. Colloids Surf. 1983, 6, 1. (34) Maeda, Y.; Hachisu, S. Colloids Surf. 1983, 7, 357. (35) Maeda, H.; Maeda, Y. Langmuir 1996, 12, 1446. (36) Onsager, L. Phys. Rev. 1942, 62, 558. (37) Onsager, L. Ann. N. Y. Acad. Sci. 1949, 51, 627.
Figure 1. (a) Polarizing microscopy image of a 50 mg/mL TMV suspension after phase separation with visible spindle-shaped tactiods, and (b) polarizing microscopy image, taken with a sensitive color plate, of oriented spindles and their fused bodies. The colors depend on the orientations of the tactoids. (Bar, top left, in part a is 100 µm, and bar, lower left, in part b is 50 µm.) sample surface, and “error signal mode” imaging, which gives differential images with respective to the height of the sample surface. In conventional contact mode AFM, the probe tip is dragged across the sample surface to obtain topographic images of the surfaces. The dragging motion of the probe, combined with lateral frictional and adhesive forces between the tip and the surface, can damage samples and distort image data. However, tapping mode AFM overcomes such problems using a vertically oscillating probe because a sufficiently large oscillation amplitude of the tip (more than 20 nm) can overcome the tip-sample adhesion forces and the resulting intermittent contact of the tip with the surface eliminates the lateral friction forces.
Results and Discussion 1. Observations by Polarizing Microscopy. Dried assemblies of the TMV particles on glass substrates showed concentration-dependent characteristic patterns, which were found to be based on the anisodimensional (or rodlike) shape and semiflexibility of the individual particles. A polarizing microscopy image of a TMV suspension at a concentration of 50 mg/mL is shown in Figure 1a. Many bright and dark spindles with sizes varying from 15 to 50 µm appeared on drying. The orientations of the bright and dark spindles are different by 90°, indicating that the spindles are birefringent, and thus, the particles in each spindle are expected to be highly oriented. The internal structure of the 2D-spindles is shown in the AFM images
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in Figure 10b,c. The spindles, called “tactoids” by Freundlich,38 have been observed in some colloidal systems including not only TMV but also other anisodimensional particles39,40 such as iron hydroxide, vanadium pentoxide, and tungsten trioxide. Bernal et al.27 explained the shape of the tactoids by postulating two different principal surface tensions, one parallel to and the other perpendicular to the direction of the particles. The tactoids are the concentrated phase of the TMV particles in the dilute phase. Onsager considered the phase separation in hard rod systems as a pure entropy effect.36,37 In Figure 1b, the oriented tactoids of TMV and their fused bodies are visible. This indicates that the tactoids are deformable, fusionable, and changeable in orientation. The characteristic patterns appearing in the dried assemblies could be caused by such characteristics (together with particle flexibility, as will be shown in Figures 8 and 9). The pattern formation process from a suspension (with a TMV concentration of 50 mg/mL) was observed during drying using a polarizing microscope with a sensitive color plate. The process was divided into three characteristic stages of formation: (1) At an early stage of the process, the oriented tactoids (blue-colored small spindles) and their fused bodies (blue- and yellow-colored domains) were observed (Figure 2a). The particle orientation in the yellow-colored region was approximately different by 90° from that in the blue-colored region. It should be noted that the sizes of the fused bodies were much larger than those of the individual spindles, showing how a wide range of highly oriented regions of the TMV particles were formed in the dried assemblies (Figure 7a,b). In this stage the suspension was still fluid. (2) In the intermediate stage of the process, the suspension began to dry from the left side (Figure 2b), and many parallel curved lines appeared. From the AFM images, each curved line was found to be a crack with a width of approximately 1 µm (see Figure 6a,b) The right side of the image was still fluid, and the fused tactoids (the blue- and yellow-colored domains like clouds) were visible. (3) In the final stage, vaporization from the suspension was completed, and the birefringent crack patterns covered all of the suspension (Figure 2c). The blue-colored regions were oriented by approximately 90° from the yellow regions. From the AFM images, the TMV particles were found to orient along the crack lines (as shown in the top right of Figure 8a). The straight, slightly or sharply curved, and broken parallel crack lines were visible, which were locally parallel over an area of at least 200 µm × 200 µm. Characteristic crack patterns are shown in Figure 3a-d using polarizing and unpolarizing microscopes. A wide range of highly oriented regions of the TMV particles is evident in these figures (see also the AFM images in Figure 7a,b). Thin lines (short arrow) perpendicular to the parallel crack lines (long arrow) are visible in Figure 3a. The perpendicular thin lines were also found to be cracks due to transverse tearing of the particles (see Figure 6c). The typical distances between the parallel crack lines, and those between the thin lines are 10-20 and 20-200 µm, respectively. Several different defects were also observed in the crack patterns. In fact, a singular point was observed at which the crack lines gather and abruptly end (as indicated by an arrow in the center of Figure 3b). This type of crack pattern is also visible in Figure 5c. In addition, another defect was observed in which three distinct crack domains are in contact with one another (38) Freundlich, H. Kapillarechemie, II, Leipzig 1932, p 55. (39) Zocher, H. Z. Anorg. Allgem. Chem. 1925, 147, 91. (40) Zocher, H.; Heller, W. Z. Anorg. Allgem. Chem. 1930, 186, 75.
Maeda
Figure 2. Polarizing microscopy image, taken with a sensitive color plate, of the formation process of the patterns in the TMV suspension on a glass substrate: (a) at an early stage, tactoids and their fused bodies are visible; (b) at an intermediate stage, parallel curved crack lines appeared in the left side of the TMV suspension; (c) finally, the parallel crack pattern covered the entire surface. These images are of different regions of the suspension. Blue- and yellow-colored domains correspond to the dark and bright ones shown in Figure 1a, respectively. (Bar, top left, in part a is 100 µm, and bars, lower left, in parts b and c are 200 µm long.)
(Figure 3c,d, taken without and with a sensitive color plate, respectively). This defect is similar to a screw disinclination, which was observed in TMV nematics using freeze fracture electron microscopy by Zasadginski et al.32 They found that the particles were much more disordered and seemed to twist in the core of the defect with a width of several particle lengths. In this study, it was determined from an AFM image of the defect in Figure 3c that the core is 1.5-2 particle lengths; the density of the particles is fairly low and the particles were randomly oriented.
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Figure 3. Optical microscopy images of dried TMV assemblies: (a) parallel crack pattern in which parallel crack lines (long arrow) and thin crack lines (short arrow) perpendicular to them are visible, (b) a cusp-like defect in a crack pattern in which the crack lines abruptly end (at the position indicated by an arrow), (c) defect in a crack pattern in which three distinct crack domains are in contact with one another around a core region, and (d) the same pattern as part c, taken with a sensitive color plate. This texture is similar to a screw disinclination observed in thermotropic nematics. (Bar, top left, is 100 µm.)
Figure 4a-c are the polarizing microscopy images (taken using a sensitive color plate) of the zigzag patterns; the zigzag lines run from the lower left to the top right. Corresponding to an alternative directional change in the zigzag lines, blue- and yellow-colored bands appeared alternatively; in each band, the broken segments of the zigzag lines oriented to almost the same direction. The average separation between the zigzag lines was 10-20 µm; the widths of the blue- and yellow-colored bands were typically 10 and 30 µm, respectively. The particle arrangement in the zigzag pattern was determined using AFM as shown in Figure 9a,b. In Figure 4a,b, two boundary lines are visible, at which the directions of the crack lines (i.e., the particle orientations) discontinuously change. In Figure 4c, the gradual variations of the segment lengths are clearly visible in each band. Dried assemblies of TMV at a concentration of 5 mg/mL also showed characteristic crack patterns, which were divided into four regions, I, II, III, and IV, from the periphery to the center of the pattern because the droplet of the suspension begins to dry from the peripheral to central regions on the glass substrate. In region I, the crack lines were parallel to the peripheral line of the suspension, and the average distance between the crack lines was 5 µm as shown in the upper side of Figure 5a. It is of interest that the particles align parallel (not vertical)
to the peripheral line. In region II, the parallel pattern began to fluctuate as shown in the lower side of Figure 5a; and in region III, a zigzag-like crack pattern with a lateral periodic distance of 30-40 µm appeared (Figure 5b). Finally, in region IV (the central region), a cusp-like pattern appeared as shown in Figure 5c. The crack lines abruptly ended at the cusp. The appearance of the different patterns (regions I-IV) suggests spatially nonuniform inward speeds of the peripheral line of the suspension droplet during drying, which could be related to the local viscosity (or mobility) of the suspension. 2. Observations by AFM. Using AFM, the parallel curved lines in the dried assemblies of TMV (Figures 2-5) were found to be cracks with a typical width of 1 µm. In fact, the light microscope showed many wedge-shaped and curved lines in Figure 5b. The AFM images for such regions also showed the wedge-shaped and curved cracks as seen in parts a and b of Figure 6, respectively. Many stream lines were also observed along the cracks; using a more extended AFM image of Figure 6b, the individual TMV particles were found to orient along the stream lines. Thin cracks perpendicular to the stream lines were frequently observed (Figure 6a,b). An extended AFM image of a thin crack showed that the parallel aligned particles are torn at the crack (Figure 6c). The lengths
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Figure 4. Polarizing microscopy images, taken with a sensitive color plate, of zigzag crack patterns. The zigzag crack lines are running from lower left to top right. Alternative blue- and yellow-colored bands are also visible, which correspond to the alternative directional changes in the (broken) segments. In each band, the segments of the zigzag lines orient in approximately the same direction. In the images of parts a and b, there are two boundary lines in which the orientations of the zigzag domains abruptly change; in part c the gradual variations of the band widths in a zigzag pattern are shown. (Bars, lower right, in parts a, b, and c are 100, 100, and 50 µm, respectively.)
of the torn particles are disperse, indicating that any of the particle sites are equally likely to be affected by external forces. In regions having the straight crack lines (Figure 3a), the TMV particles were found to be highly oriented over a wide range as shown in the AFM images in Figure 7a,b; the distance between the parallel lines coincides with the average diameter of the TMV particle, dave (see the next section), and the small dark rectangular parts are gaps between the ends of the neighboring particles. From the AFM images of the highly oriented regions, the size of the
Figure 5. Polarizing microscopy images for dried assemblies of a 5 mg/mL TMV suspension: (a) parallel crack pattern of the peripheral region, (b) zigzag-like crack pattern with a lateral periodic distance of 30-40 µm, (c) cusp-like pattern in the central region. (Bars, lower right, in parts a, b, and c are 50, 50, and 100 µm, respectively.)
TMV particles can be determined. A longitudinal height profile (i.e., a cross section along the long axis) of the particles in the highly oriented region (Figure 7a) is shown in Figure 7c. It should be noted that the scale of the height is extremely enlarged compared with that of the length.
Assembly Structures of Tobacco Mosaic Virus
Figure 6. AFM (error signal mode) images for dried assemblies of TMV: (a) wedge-shaped and (b) curved cracks with visible stream lines along the cracks in each image, and (c) a crack that is perpendicular to the parallel particles. All the TMV particles are torn. The images of parts a and b correspond to the zigzag-like domain and a curved domain just below the zigzag-like domain, respectively.
Sharp valleys (or gaps between the ends of neighboring particles) are visible at around 100 and 400 nm. The
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particle length (i.e., the distance between the two arrows at 103.6 and 404.1 nm in the figure) is approximately 301 nm. There have been many reports on the length of the TMV particles using electron microscopy (listed in Table 1 as EM). The published values of the particle length are in good agreement with ours, obtained by AFM. (However, the particles longer than 300 nm were frequently observed in the oriented regions as shown in Figure 7a,b. This unambiguosly indicates the interparticle end-to-end connection.) The height profile curve further shows the roughness of the particle surface; the height difference between the ridges and grooves of the surface is approximately 0.47 nm. Figure 7d is a transverse height profile (i.e., a cross section perpendicular to the long axis) of the TMV particles shown in Figure 7b; the cross section of 12 parallel particles gives an average particle width of 14.7 nm (denoted by dave in Table 1). Bernal et al.27 estimated a value of 15.2 nm for the interparticle distance in a close-packed gel of TMV using X-ray diffraction techniques. Matthews et al.28 also obtained the particle width as 15 ( 0.5 nm in a close-packed region using electron microscopy. These values for the particle width are in close agreement with ours. An error signal mode AFM image (Figure 8a) shows an orientational transition of the TMV particles in the corner of a curved region between the parallel and zigzag-like domains (Figure 5a,b). In the outer part of the curved corner, the individual particles are slightly bent and continuously change their directions along the peripheral curved (crack) line (as shown in the top right of Figure 8a). In the inner part from a critical point (indicated by an arrow) the continuously curved corner changes to a deflected corner such that the particles discontinuously turn their orientations by 80° (without bending) at a line that ends at the critical point. This transition indicates the existence of a critical bending angle of the individual particles. The particles bent by 60° (a deviation angle from the long axis of the particle) were observed in a curved region (Figure 8b); the bending angle of 60° was estimated by applying a 3D-analysis to the bent particles in the AFM image. The bending angle of 60° was more exactly observed in the zigzag domain shown in Figure 4a-c; the parallel particles were observed to be bent at a deflection line (Figure 9a). An extended AFM image of a region arround the position indicated by an arrow in Figure 9a showed the 60°-bent particles more clearly (Figure 9b), but broken particles were also observed at the deflection line, suggesting that the angle of 60° is a critical bending angle. The bending of the particles mentioned above can be explained as follows: The particle consists of protein subunits in a helical aggregate; each of the subunits has six nearest neighbors or six bonds. A mean free energy change per bond has been estimated to be -2.7 kcal/mol.18 This bond energy represents the sum of all of the interactions (such as hydrogen bonds, salt links, and hydrophobic bonds) between a neighboring pair of the subunits in the helix. The mean free energy is only about five times larger than the average kinetic energy of random thermal motion (approximately 0.6 kcal/mol). Many molecules with sufficient kinetic energy to break the weak bond always exist at physiological temperature (because there is a significant spread in the energies of kinetic motion), but the helix is nevertheless stable in the suspensions. The stability is due to the coordinated arrangement of a large number of relatively weak interactions in an ordered structure. In the highly concentrated suspensions, however, macroscopically systematic (not random) forces are expected to occur on drying, since the various macroscopic shapes of the
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Figure 7. AFM images of a highly oriented region of TMV particles. In parts a and b, all the particles are uniaxially oriented but longitudinally random; small dark parts are voids (gaps) between the ends of the neighboring particles. The width of the particles (14.7 nm) corresponds to the average diameter of TMV particles determined by X-ray analysis. (c) Longitudinal and (d) transverse height profiles of the TMV particles in parts a and b of Figure 7, respectively.
assembly patterns appear on the substrates. The systematic bending and tensile forces acting on the assemblies consisting of the highly oriented particles can generate sufficient local forces to overcome the weak bond interactions and to expand the inter-subunit distances, resulting
in bending and tearing of the individual particles in the dried assemblies. In the curved (Figure 8a,b) and zigzag (Figure 9a,b) assemblies, the interparticle end-to-end connections were also observed as well as in the highly oriented regions
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Table 1. Particle Sizes of TMV Obtained by Various Methods (in nm)a length AFM*3 AFM*2 AFM*7 AFM*8 AFM*4 AFM*6 AFM*5 AFM# STM42 STM7 STM43 X-ray22 X-ray EM14 EM16 EM EM EM EM
dave
dmax
300 300 290
300 ( 10 301 ( 2 300 290
14.7
dapp
hmax
48.3 ( 3.1 (Wb) 100-150 (Wb) 25 67.6 ( 12.4 45 ( 21, 60, 28 (Wm) 82.2 ( 10.1 (Wb) 30 ( 10 47.7 (Wb), 32.6 (Wm) 70-100 (Wm)
17.8 14 19 14.9 ( 3.4 9 ( 0.5, 5, 7 19.3 ( 1.2 8(3 16.8-18.6 20 12 9-14
30< 15.4 15.2 27 300 287 280-300 11 298 ( 1 12 295-305 13 289 ( 1 15
15.0 ( 0.5 28
18 18 24 18.5 18-20 18 25,26
a The superscript numbers indicate the reference numbers. AFM*: contact mode AFM. AFM#: tapping mode AFM (present work). STM: scanning tunneling microscopy. EM: electron microscopy. dave: average width of a TMV particle intermeshed in its close-packed array. dmax: maximum width of an isolated TMV particle. dapp: apparent width of a TMV particle due to the finite size of the probe tip. hmax: maximum height of a TMV particle lying on a substrate. Wm,Wb: apparent middle and bottom widths of transverse height profiles of the single TMV particle, respectively.
(Figure 7a,b) because the gaps between the ends of the neighboring particles frequently were not observed. Such a connection seems to be reasonable, considering the weak bond energy between the protein subunits. The interparticle separations in the curved and zigzag assemblies (Figures 8 and 9) were determined from the height profiles to be 16.4 and 17.2-29.0 nm, respectively. These values are larger than those of the intermeshed particles, suggesting that the intermeshed state is formed particularly in the highly oriented but undeformed, dried assemblies. At an extremely low concentration of TMV, the characteristic 2D-network patterns appeared (Figure 10a), which are formed by connecting thin spindles with other ones using their own multibranches. In the network, the 2D-spindles and -bundles and their fusing and branching structures are visible (Figure 10a). From more detailed observations of the images, the formation of the multibranching of the particle seems to be due to an anisodimensional (or rodlike) shape, semiflexibility of the particles, and deformability of the spindles. The bundles (0.5-1.5 µm long) were formed by 2-10 laterally aligned particles and were longitudinally random; the interparticle separations were approximately 16.7 nm, indicating that these particles were also not intermeshed with one another. In the regions where the edge of the suspension droplet moved slowly, the dense network patterns of the spindles appeared, while in the regions where the edge moved quickly the dilute network patterns of the thin bundles appeared. This suggests that the numbers and sizes of the spindles increase with increasing drying time. Furthermore, an extended AFM image (Figure 10c) showed that the particles in the outer region of each spindle are slightly bent and gradually change their orientations, and the densities of the inner particles decrease toward both ends. In the spindles (0.5-1 µm long), the particles were also observed to be longitudinally random. WaduMesthrige et al.5 also found similar 2D-network patterns from low-ionic suspensions of TMV on mica and albumincoated mica. This suggests that the presence of the ions has little effect, at least, on the 2D-network structures on the substrates. Further, they found that the network patterns were not formed in the TMV/albumin systems and suggested that interparticle and/or particle-substrate
interactions can be changed by the presence of albumin. Also, the preferential adsorption of TMV on a mixed hydrocarbon and fluorocarbon LB film was observed by Frommer et al.9,10 3. Evaluation of Particle Size and Probe Tip Geometry. From X-ray diffraction measurements, Franklin et al.22 showed that the TMV particle was not a cylinder but a rod bearing a helical groove; the depth of the groove is approximately 3 nm and the maximum diameter (dmax) was about 18 nm. Using cryo-electron microscopy similar values for dmax were reported.25,26 Franklin et al.22 further showed that the helices of the neighboring particles can intermesh with one another in the close-packed gels, resulting in an interparticle separation of 15.4 nm. This value agrees well with the particle width in the highly oriented region, 14.7 nm by AFM in this work (Figure 7a,b). To evaluate the sizes (length, width, and height) of the single (i.e., non- intermeshed) particle, and to evaluate the tip geometry effect on the height profiles, we used the particle images of the 2D-networks of Figure 10a. The longitudinal height profile (i.e., the cross section along the long axis) of a single particle seen in Figure 10a is shown in Figure 11a. The profile width was distorted so as to be wider toward the bottom; the particle lengths at the bottom, middle, and top of the profile were approximately 318, 304, and 291 nm, respectively. On the contrary, the length of the particles were almost uniquely determined to be 301 ( 2 nm in the highly oriented regions of Figure 7a,b. The distortion of the image is obviously attributable to the finite size of the probe tip. The transverse height profiles (i.e., the cross sections perpendicular to the long axis) of two different particles, one whose ends are fixed by bonding to neighboring particles and the other which is free from others, are shown in parts b and c of Figure 11, respectively. The transverse section of the TMV particle must be a circle, but the observed sections were distorted so as to be wider toward the bottom as follows: (1) in Figure 11b the particle widths at the bottom and middle are 47.7 and 32.6 nm, respectively, and (2) in Figure 11c they are 65.1 and 45.8 nm, respectively (the former values are listed in Table1 as dapp). One of the causes for the broader apparent widths of the free particle is the displacement of the particle by the scanning tip. To evaluate these experimental values
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Figure 8. (a) AFM (error signal mode) image of the corner of a curved region between the parallel and zigzag-like regions (Figure 5a,b). The individual TMV particles are visible. At the outer region of the corner, the individual particles gradually change their directions with slight bending, while at the inner region from a critical point (an arrow) the particle orientations discontinuously change by 80° without bending. This suggests the existence of a critical bending angle for the individual particles. (b) Extended AFM image of the bent particles in the curved region.
for the widths, which are significantly larger than the dmax determined by Franklin et al.,22 a theoretical height profile for the particle (i.e., the trace of the tip apex which closely contacts with the particle surface during scanning in “contact mode” AFM) is needed. The widths at the bottom and middle heights of the profile curve (denoted by Wb and Wm, respectively, which are illustrated in Figure 12) can be easily calculated as follows
Wb ) 2{(r + R) + (r - R) sin θ}/cos θ for sin θ > (R - r)/(R + r) (1) ) 4 xRr for sin θ < (R - r)/(R + r)
(1′)
Figure 9. (a) AFM image of the zigzag pattern region, and (b) tilted and enlarged AFM image of the sharply bent particles. In part a, the parallel particles are sharply bent at a deflection line. An arrow indicates the position which coincides with the center of the zoomed area shown in part b. In part b the bending angle (or the deviation angle from the long axis) was determined to be approximately 60°.
Wm ) 2{R(1 - sin θ) + r}/cos θ for sin θ > R/(R + r) (2) ) 2xr(r + 2R) for sin θ < R/(R + r)
(2′)
where r is the radius of a specimen, R is the radius of the tip apex, and 2θ is the cone angle of the tip. If the theoretical height profile for the contact mode can be used even for the tapping mode, we can calculate R using eqs 1-2′. If r ) 9 nm (for TMV), and R ) 5-10 nm and 2θ ) 35° (these are the nominal values for a commercial probe tip), eqs 1 and 2′ should be used for calculating R. If Wm ) 32.6 nm (a measured value for the fixed single particle) and r ) 9 nm (for TMV) are substituted into eq 2′, one can obtain R ) 10.3 nm. Similarly, if Wb ) 47 nm, r ) 9 nm, and 2θ ) 35° are substituted into eq 1, we obtain R ) 12.7
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Figure 11. (a) Longitudinal height profile of a TMV particle in a 2D-network structure, and transverse height profiles of TMV particles with (b) one bonded to neighboring particles and (c) the other free from the others. Both transverse height profiles are distorted so as to be wider toward the bottom due to a finite size of the probe tip.
the size of TMV to be 48.3 nm wide (Wb) and 17.8 nm high, using contact mode AFM. They estimated the radii of the commercially available and lithographically sharpened probe tips that they used to be 10.9 and 9 nm, respectively, using a paraboloidal tip model. In the theoretical height profile (illustrated in Figure 12), ht, which indicates a height giving a true particle width (2r) can be obtained as follows
ht ) r - R + xR(R + 2r)
Figure 10. (a) AFM (error signal mode) image of a 2D-network pattern of the TMV particles, (b) the arrangement of the TMV particles in the network, and (c) extended AFM (error signal mode) image of the fused spindles. The spindles (or bundles) and their fusing and branching structures are visible in part b. The arrangement of the particles are clearly visible in part c.
nm. These values of R coincide well with the maximum value of the nominal radius for the tip apex. However, Wm (eq 2′) is more suitable for evaluating the radius of the tip apex, because it has only R. Thundat et al.3 determined
(3)
where R and r have the same definition as in eqs 1 and 2. When r ) 9 nm (for TMV) and R ) 10.3 nm (estimated above) are substituted into eq 3, the value of ht/hmax (hmax is the maximum height in the profile curve or 2r) determined is 0.88. This value is in good agreement with the observed value, 0.87. Thus, this is a convenient method for obtaining the true particle width from the observed height profile. We further tried to measure the depths of the valleys between the intermeshed particles or the separated ones observed in the highly oriented region (Figure 7a) using tapping mode AFM. The depths of the periodic valleys between the intermeshed particles are shown to be 0.81.5 nm in Figure 7d. Furthermore, the transverse and longitudinal depth profiles of the gaps having one particle width (e.g., a thin void in the highly oriented region of Figure 7a) are shown in parts a and b of Figure 13, respectively. The maximum depths are 4.8 nm for the transverse profile (Figure 13a), and 5.5 nm for the
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Figure 12. The theoretical height profile (solid line) in the contact mode is illustrated, together with a probe tip (with an apex radius R) and the TMV particle (with a radius r). In the profile hmax is the maximum height of the profile, and rt is the height giving the particle width. Wb and Wm are the widths at the bottom and middle of the profile, respectively.
Figure 14. Height profiles of multialigned and single-, two-, and three-aligned, laterally connected TMV particles. The sections are not necessarily perpendicular to the long axis of the respective particles.
Figure 13. The observed depths for (a) transverse and (b) longitudinal profiles of gaps having one particle width are 3.64.8 nm and 3.7-5.5 nm, respectively. The theoretical maximum depth, Dmax, of the gap between two TMV particles with a width of 2W is illustrated: (c) the particles are intermeshed particles, and (d) the particles are separated by one particle width.
longitudinal profile (Figure 13b). To evaluate these values, the maximum depths (Dmax) with a particle separation of 2W (as illustrated in Figure 13c,d) can be expressed by the following equations
Dmax ) (R + r)(1 - 1/sin θ) + W/tan θ for (R + r) cos θ < W (4) ) (R + r) - x(R + r)2 - W2 for (R + r) cos θ > W (4′) where R, r, and θ have the same definitions as in eqs 1-2′. When R ) 10.3 nm (estimated above), 2θ ) 35°, and 2W ) 15 nm (for the intermeshed particles), eq 4′ must be used (because only the condition in eq 4′ is fulfilled), which gives Dmax ) 1.5 nm, while from Figure 7d the experimental values of Dmax were 0.8-1.5 nm. Furthermore, when R ) 10.3 nm and W ) 15 nm (for a void having one particle width, e.g., it is visible in Figure 7a), eq 4′ gives Dmax )
7.2 nm, while the experimental Dmax was 4.8-5.5 nm (Figure 13a,b). These discrepancies between the experimental and theoretical values of Dmax could be due to a response delay of the piezoelectric scanner on which the samples were mounted. (The scanner was operated so as to quickly follow the height change of the surface and to keep the tapping amplitudes of the cantilevers constant during scanning.) To obtain dmax for the TMV particle, the height of the particle lying on the substrates can be used, because the values of width and height are the same for the cylindrical particle. Figure 14 contains examples of the height profiles for the single-, two-, and three-aligned and multialigned, laterally connected particles in Figure 10a (the cross sections are not necessarily perpendicular to the long axis of the particles). In particular, for the single-, two-, and multi (i.e., > 3)-aligned particles, the histograms for the occurrence vs particle height are obtained (Figure 15); the mean height of each is 18.6, 17.4, and 16.8 nm, respectively. Considering that dmax is 18 nm and the groove depth is 3 nm (Franklin et al.22), hmax and hmin, which denote the heights of the top and bottom of the groove of the particle lying on the substrate from the substrate surface, respectively, are 18 and 15 nm, respectively. Consequently, the height observed for a single particle lying on the substrate gave almost the same value as dmax (18 nm), and that for the multialigned particles gave approximately the mean of hmax and hmin. As shown in Table 1, the particle lengths determined by contact mode AFM are in good agreement with ours observed using tapping mode AFM; however, the heights determined by the contact mode are frequently smaller than ours. This seems to be due to particle deformation, suggesting that, in the contact mode, the interactions
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molecules in their native states and, additionally, eliminate the contamination layers on the sample surfaces which are formed in the presence of air. This suggests the possibility of obtaining higher resolutions with biological molecules. In fact, the observations for TMV in 2-propanol8 and 2-butanol5 have been reported. However, tight sample fixation without any denaturation is still an important problem, particularly in physiological environments.
Figure 15. Histograms for the occurrence vs particle height of single-, two-, and multi (i.e., > 3)-aligned particles which appeared in the 2D-network patterns in Figure 10a. From these histograms, the average height is estimated to be 18.6, 17.6, and 16.8 nm, respectively, for the aligned particles.
between the tip apex and particle are so strong as to displace and deform the particles (when compared to those in the tapping mode). Such an effect is particularly serious for soft substances like biomolecules. Bustamante et al.41 measured the heights and the widths of DNA molecules using contact mode AFM and obtained heights of 0.651.89 nm, although the true diameter of DNA is approximately 2 nm. This discrepancy represents distortion of the DNA molecules by the probe tip. A similar situation also occured in scanning tunneling microscopy (STM) measurements for the TMV particles. The particle sizes determined by STM7,42,43 are shown in Table 1. The disadvantages inherent in the contact mode measurements are largely obviated in the tapping mode by eliminating lateral friction forces that can damage samples and distort image data. However, the deconvolution techniques for AFM images44-46 and sample fixation are needed to obtain good images of isolated molecules. Taking into account this point, high-ordering techniques, particularly for biomolecules, are of great importance because in the ordered structures only the tip apex can interact with the particle surfaces, and so no influence of the tip shape is evident in the images. In addition, the atmosphere surrounding biological molecules is important for obtaining good AFM images. In particular, the presence of liquids can keep the (41) Bustamante, C.; Vesenka, J.; Tang, C. L.; Rees, W.; Guthold, M.; Keller, R. Biochemistry 1992, 31, 22. (42) Mantovany, J. G.; Allison, D. P.; Warmack, R. J.; Ferrell, T. L.; Ford, J. R.; Manos, R. E.; Thompson, J. R.; Reddick, B. B.; Jacobson, J. B. J. Microsc. 1990, 138, 109. (43) Guckenberger, R.; Arce, F. T.; Hillebrand, A.; Hartmann, T. J. Vac. Sci. Technol. 1994, B12 (3), 1508. (44) Keller, D. Surf. Sci. 1991, 253, 353. (45) Odin, C.; Aime´, J. P.; Kaakour, Z. E.; Buhacina, T. Surf. Sci. 1994, 317, 321. (46) Markiewucz, P.; Goh, M. C. Langmuir 1994, 10, 5.
Summary Dried assemblies of tobacco mosaic virus on glass substrates showed concentration-dependent characteristic patterns. The pattern formation process during drying was examined using polarizing microscopy, including tactoid formation, tactoid fusing, and crack pattern formation. The arrangements of the TMV particles in the patterns were investigated using atomic force microscopy. In the dried assemblies of TMV (at a concentration of 50 mg/mL), uniaxially oriented and zigzag pattern domains appeared over a wide area of approximately 200 µm × 200 µm. In the uniaxially oriented domains, the particle width and length were determined to be 14.7 and 301 nm, respectively, which agree well with published values obtained by other methods. It has been demonstrated that the highly oriented assemblies of TMV can be used as a standard specimen in the scale of 10 -100 nm. In the zigzag pattern domains, sharply bent particles were observed; a critical bending angle of 60° (a deviation angle from the long axis of the particle) was found, indicating that TMV is a semiflexible rod rather than a hard rod. In these domains, the end-to-end connections between the neighboring particles were frequently observed. Some types of defects were observed in the crack patterns: (1) a line defect, at which the particle orientations discontinuously change; (2) a screw disinclination, which has a core width of 1.5-2 particles width; and (3) a cusp-like defect, in which the crack lines (along which the particles are oriented) ended abruptly. For a TMV concentration of 0.5 mg/mL, the characteristic 2D-networks appeared in the dried assemblies. The network structures were found to be formed by branching and fusing of the 2D-spindles of TMV. The particle arrangements in the spindles were also observed by AFM. It has been shown that the radius of the tip apex of the probe can be determined from an apparent width of a single particle in the 2D-network structures. The measured heights of the particles in the 2D-networks were determined to be 16.8-18.6 nm, showing that the particles are not crushed by the tapping probe tips. Acknowledgment. We thank Drs. C. Masuda and M. Suzuki for providing us with TMV particles and Dr. K. Namba for useful discussions. This work, partly supported by NEDO, was performed in the Joint Research Center for Atom Technology (JRCAT) under the joint research agreement between the National Institute for Advanced Interdisciplinary Research (NAIR) and the Angstrom Technology Partnership (ATP). LA962105E
