Langmuir 1993,9, 2281-2288
2281
Probing the Resolution Limits and Tip Interactions of Atomic Force Microscopy in the Study of Globular Proteins Steven J. Eppell,? Fredy R. Zypman: and Roger E. Marchant'pt Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106, and Department of Physics and Electronics, University of Puerto Rico, Humacao, Puerto Rico 00791 Received October 14,1992. In Final Form: June 17,199P It is suggested that the atomic force microscope, AFM, operating in repulsive force mode with an insulatingtip, is sensitiveto charged objects on an insulatingsurface. Thia sensitivity is shown to significantly affect the topographical images obtained. Theoreticalcalculationsindicate that an electrostaticattractive force as large as 7.7 nN exists between a charged polystyrene sphere on mica and the AFM tip. Thie result is consistent with the experimental measurements obtained. Globular domains of a blood plasma glycoprotein, von Willebrand factor, were also measured using the AFM to have elliptical cross sections with major axis = 106 (f22) nm and minor axis = 81 (f22) nm and heights of 3.4 (fQ.83)nm. These domains were modeled with the 14 nm diameter polystyrene spheres. This approach allowed us to measure the instrument responseand accountforlateral image distortiondue to tip sizethus enhancingthe resolution of our data. Introduction The importance of understanding the interactions of proteins with different solid surfaces has generated considerable activity from a broad section of the scientific community. Our interest in this field is with globular proteins such as fibrinogen and von Willebrand factor (vWF), that play a significant role in thrombus formation and cell adhesion on biological and synthetic biomaterial surfaces. The complex structure of proteins, coupled with their sensitivity to small perturbations in environmental conditions, has made elucidation of their behavior on surfaces a formidable challenge.' The atomic force microscope (AFM)? invented in 1986, offered high hopes of providing access to unique insights into three-dimensional protein structure and protein-surface interactions. There were early indications that biopolymer interactions could be viewed in real time in situ with molecular scale resolution? DNA was imaged in saline with molecular scale resolution4 and there have been several studies on imaging proteins with the AF'M."' Our past work with AFM focused on vWF.8 This protein is particularly important in maintaining hemostasis under high shear conditions by binding blood platelets to the
* To whom correspondence should be addressed. + Case Westem Reserve University.
University of Puerto Rico.
* Abstract publiihed in Advance ACS A bstracts, Auguat 15,1993.
53 nm
27 nm I
Figure 1. This f i e combineswhat is known from TEMstudiea on vWF with our present understanding of imaging by AFM. Shown at the bottom is a scale diagram of the vWFpromoter as determined by TEM 26 nm globular domains (GG), 34 nm fibrillar domains(RR),and a 5 nm nodule located at the symmetry point of the protomer. GG and FtR domains are shown as being 3 nm tall. If the radius of curvature of the AFM tip were large enough,it would inhibit the tip from probing the fibrillar domains of theproteine and it would distort the sizeof the globular domains so that their diameters would appear increased as shown by simulated AFM profiie at top. Dimensions of the AFM profiie (determined by assuming a spherical tip with radius of 14 nm and a globular domain height of 3.3 nm) are shown.
(1) (a) Giroux, T. A.; Cooper, 5. L. J. Colloid Interface Sci. 1990,139, surface of a damaged blood vessel and releasing Factor 361. (b) Lewandoweka, K.; Kaetzcel, C. S.;Zardi, L.; Culp, L. A. FEBS VIII. While this scenariois consistent with the literature: Lett. 1988,237,35. (c) Chuang, H. Y. In Blood Compatability; Williams, D. F., Ed.; CRC Press: Boca Raton, FL, 1987; Vol. 1, p 87. (d) Andrade, it has never been observed microscopically. Transmission J.D. InSurfaceandInterfacial Aspects of BiomedicalPolymers: Protein electron microscopy (TEM) studies of VWF'O-~~ did not Adsorption; Andrade, J. D., Ed.;Plenum Press: New York, 1986; Vol. 2, result in easy visualization of the protein on a submolecular P 1. (2) Binnig, G.;Quate, C. F.; Gerber, Ch. Phys. Rev. Lett. 1986,56,930. scale but did provide evidence that the structure of the (3) Drake,B.;Prater,C.B.;Weisenhom,A.L.;Gould,S.A.C.;Albrecht, molecule is as shown in Figure 1. Thus, the repeating T. R.; Quate, C. F.; Cannell, D. 5.;Hansma, H. G.; Hansma, P. K. Science 1986,243,1686. (9) Ruggeri, 2. M. Mayo Clin. Proc. 1991,66, 847. Lollar, P. Mayo (4) Weieenhom, A. L.; Egger, M.; Ohnesorge, F. Langmuir 1991, 7,8. Clin. Proc. 1991, 66, 624. (6) Wiegrabe, W.; Nonenmacher, M.; Guckenberger, R.; Wolter, 0.J . (10) Fowler, W. E.; Fretto, L. J.; Hamilton, K. K.; Erickson, H. P.; Microsc. (Ozford) 1991,163,79. McKee, P. A. J. Clin. Invest. 1985, 76, 1481. (6) Butt, H.; Downing, K. H.; Hansma, P. K. Biophys. J. 1990, 58, (11) Fretto, L. J.; Fowler, W. E.; McCaalin, D. R.; Erickson, H. P.; 1473. McKee, P. A. J. Biol. Chem. 1986,261, 16679. (7) Egger, 0.M.; Ohnesorge,F.; Weisenhorn,A. L.; Heyn, S.P.;Drake, (12)0hmori, K.; Fretto, L. J.; Harrison, R. L.; Switzer, M. E. P.; B.; Prater, C. B.; Gould, S. A. C.; Hausma, P. K.; Gaub, H. E. J. S t r u t . Erickson, H. P.; McKee, P. K. A. J. Cell Biol. 1982,96,632. B 1990,103,89. (13) Slayter, H.; Loscalzo, J.; Bockenstedt, P.; Handin, R. I. J. Biol. (8).Marchant,R. E.; Lea, S. A.; Andrade, J. D.; Bockenstedt, P. J. Chem. 1985,260, 8569. Colloid Interface SCL1992, 148, 261.
0 1993 American Chemical Society
Eppell et al.
2282 Langmuir, Vol. 9,No. 9,1993
structure of vWF is believed to consist of three globular domains attached by flexible fibrillar domains. We expected the AFM to probe the three-dimensional structure of the molecule with enhanced resolution providing a direct measure of the molecule's height and lateral dimensions, thus leading to conclusions about the structure-function relationship of vWF on various biomaterial surfaces. Our studies of vWF on mica showed that, in the repulsive mode, the applied lateral force of the tip was sufficient to move the protein around while imaging.8 Removal of interfacial water by drying in air immobilized the protein allowinghigh-resolutionimages to be obtained. The images showed distinct globular regions along the vWF multimer consistent with what would be expected for the native molecule. However, we observed significant enlargement over the expected dimensions of the globular domains which raised questions regarding the effects of the tip-sample interactions on the images and on the resolution limits of the AFM in the study of globular proteins. A conclusion that may be drawn from past studies is that images of surfaces obtained by using an AFM are useful only if one understands the interaction between the probe tip and the sample surface. The importance of the tip-sample force issue has been appreciated by other investigators as evidenced by several publications addressing the issue from both the theoretical14and experimental15perspectives. There are theoretical publications dealing with van der Waals attractive forces and hard core repulsion due to charge overlap between tip and surface,16 as well as electrostatic Coulomb forces between tip and surface.17 Other experimental reports have described the manipulation of proteins and polymers due to an applied lateral force of the AFM tip,sJ8Jgthe effects of a capillary force due to ambient humidity when imaging and imaging localized charge with a sharp tip.20 However, there have been no studies in which a model system was used to quantify the tip-surface interactions providing information that enhanced the measurements of an unknown surface. This report elucidates the interaction between an AFM probe tip and vWF using small polystyrene spheres on mica. Our goal was to use the polystyrene spheres as models for the globular domains of vWF in order to quantify the effects of tip-surface interactions and thus improve the resolution of the information obtained by the AFM. We obtain AFM images of vWF which agree with TEM data on a 100-nm scale, but not on a 10-nm scale. By analyzing the polystyrene sphere images, we show how to measure the systematic lateral enlargement of objects (14) (a) Howells, S.; Chen, T.; Gallagher, M.; Yi, L.; Sarid, D. J. Appl. Phys. 1991, 69, 7330. (b) Tomanek, D.; Overney, G.;Miyazaki, H.; Mahanti, S. D. Phys. Rev. Lett. 1989,63,876. (15) (a) Bustamante, C.; Vesenka, J.; Tang, C1 L.; Rees, W.; Guthold, M.; Keller, R. Biochemistry 1992,31,22. (b) OShea, S. J.; Welland, M. E.; Rayment, T. Appl. Phys. Lett. 1992,60,2356. (c) Weisenhorn, A. L.; Maivald, P.; Butt, H.-J.; Hansma, P. K.Phys. Reu. B 1992,45,11226. (d) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Nature 1991,353,239. (e) Burnham, N. A.; Colton, R. J.Pollock, H. M. J. Vac. Sci. Technol., A 1991,9,2548. (0Thundat,T.; Warmack, R. J.; Allison,D. P.;Bottomley, L. A.; Lourenco, A. J.;Ferrell, T. L. J. Vac. Sci. Technol.,A 1992,10,630. (16) (a) Zhong, W.; Overney, G.;Tomanek, D. Europhys. Lett. 1991, 15,49. (b) Goodman, F. 0.; Garcia, N. Phys. Rev. B 1991,43,4728. (c) Gould, S. A. C.; Burke, K.; Hansma, P. K. Phys. Rev. B 1989,40, 5363. (d) Batra, I. P.; Ciraci, S. J. Vac. Sci. Technol., A 1988, 6, 313. (17) Bergasa, F.; Saenz, J. J. Ultramicroscopy 1992, 42, 1189. (18) Lea, A. S.; Pungor, A.; Hlady, V.; Andrade, J. D.; Herron, J. N.; Voss, E. W., Jr. Langmuir 1992,8, 68. (19) Leung, 0. M.; Goh, M. C. Science 1992,255, 64. (20) (a) Stern, J. E.;Terris, B. D.; Mamin, H. J.; Rugar, D. Appl. Phys. Lett. 1988,53, 2717. (b) Terris, B. D.; Stern, J. E.; Rugar, D.; Mamin, H. J. J. Vac. Sci. Technol., A 1990,8,374. (c) Terris, B. D.; Stern, J. E.; Rugar, D.; Mamin, H. J. Phys. Reu. Lett. 1989, 63, 2669.
by the AFM;we also quantify the effect of static charges on the interaction between the probe tip and a charged sphere. This analysis allows significant improvement in the accuracy of the measured lateral dimensions of the vWF globular domain. However, we discover a large discrepancyin the height measurement of the polystyrene spheres, which is attributed to the presence of an electrostatic interaction between the AFM tip and the sphere. We confirm the validity of this hypothesis both experimentally and theoretically. The interactions are then discussed in the context of the globular protein, vWF, but our results have direct relevance to studies on other biological and synthetic macromolecules. Experimental Section SamplePreparation. The surfaces examined with the AFM were prepared by gluing 1-cm2pieces of muscovitemica to a steel disk. Mica cleavage was begun with a sharp tungsten needle and completed with a pair of tweezers. For the vWF experiments, human vWF (prepared as described previously)8was diluted to 40pglmL in lOmM ammoniumacetate buffer, then a 2-pL aliquot was deposited on the mica. The surface was then vacuum dried for sever1hours in a sorption pumped stainless steel chamber at -1 X 10-3 Torr. Polystyrene spheres (carboxylate modified latex from IDC Corp., Portland, OR) packaged in distilled water with no surfactants, stabilizers, or preservatives were used as model structures. IDC measured the diameter of the spheres using TEM. Five hundred sphere diameters were recorded resulting in an average diameter of 14 nm (h3.7nm). The surface charge density of the spheres was measured using conductometric titration. This measurement was done twice, resulting in a mean charge density of 2.67 pC/cm2 (f0.02 pC/cm2). For theae experiments, a 20-pL aliquot of 14 nm diameter polystyrene spheres which had been diluted to 3.5 x 109 sphereslpl was deposited on the mica. The surface was then dried for several hours. To neutralize the charge on the spheres, two aliquots, 4 pL each, of Ultrex Ultrapure perchloric acid (J.T. Baker, Inc., Phillipsburg, NJ) diluted to pH 4 were deposited on the surface. During deposition,the surfacewas rotated at lo00 rpm to assist removal of the acid from the surface. Finally, the surface was vacuum dried and imaged on the same day that the untreated surface was imaged. AFM. All samples were imaged under ambient air conditions with a commercial AFM (Nanwope 11, Digital Instruments, SantaBarbera, CA) whichusesanopticallever tomeasurerelative changes in tip position. Commercial SiaN, tips integrated on SisN4 cantilevers (Nanoprobes, Digital Instruments, Santa Barbera, CA) were used. The cantilevers were -100 pm long, triangularly shaped, with a manufacturer's reported spring constant of 0.6 N/m. The tips were pyramidally shaped and -5 pm tall.Typical engagementforceswere l00nN as determined by multiplying the cantilever spring constant by the distance the cantilever was flexedbetween its set point and the point at which the tip disengaged from the surface. The AFM was calibrated with an optical diffraction grating before imaging the proteins. The spacing of the grating was imaged to within 3% of the actual spacing, lo00 nm. As a test of the linearity of the response of the AFM over a range of length scales, we measured the spacing between carbon atoms on an HOPG(bp) surface. We found that the atom spacing was always within 0.05 nm of the known 0.26 nm spacing imaged by scanning probe techniques on this surface. All AFM images reported below were taken in what is commonly referred to as the constant forcemode. In this mode of operation, a digital feedback loop uses the position of a focused h e r beam (which is reflected off the back of the cantilever and onto a split photodiode) as ita input and voltage to the z-piezo as its output to maintain a constant laser beam position (ILP). This requires that a set point position be chosen for the laser beam. We chose for this set point the position that the h e r beam assumes when the tip and sample surface are far apart (>5 pm).
-
-
Results and Discussion vWF Molecules. We began our studies by examining vWF, which had been deposited on mica. Lateral reso-
Langmuir, Vol. 9,No. 9,1993 2283
AFM Study of Globular Proteins
12.00
I I
I
-
.-_-
.
m.0 lk
u,.
1-
Iy
1. WI
vWF on Mica
Jo.O
Iy
I
- L-J
I
II.-
0."
, I)**
L, I I
vWF on Mica
I
250
I 0
Figure 2. (A, top left) This 2.5 pm X 2.5 pm image of vWF on mica is typical of the surfaces studied by AFM. The majority of the material imaged on the mica surface is present as extended chains of protein. There are several regions in which these chains overlap (aggregates) or are coiled. The "piling up" of protein in these conformations causes them to be imaged as higher (lighter brown) than the extended portions of the protein. (B, bottom left) A histogram of the heights of the pixels from part A is shown. The heights are referenced to the lowest pixel in the image. The inset has a color table chosen so that each change in color corresponds to a 3 nm change in height (in order of increasing height: yellow, green, light blue, dark blue, magenta). The peak around 2 nm corresponds to the mica substrate. The peak around 5 nm corresponds to the extended portions of the vWF. Thus, the extended vWF domains are about 3 nm above the average mica background. (C, top right) a portion of a vWF chain is shown meandering up through the center of the field of view. This protein segment is indicated by an arrow in part A. (D, bottom right) The same protein segment shown in part C is shown. Dimensions of the globular domains marked A through J as listed in Table I. The black ellipses represent the size of the globular domains after accounting for a tip radius of 14 nm. The distance d is 160 nm, in good agreement with TEM data for vWF protomers.
lution high enough to image separate globular domains within a single protein molecule was obtained. Figure 2A is a typical 2.5 pm X 2.5 pm view of the vWF on mica surfaces examined showing both aggregates of folded chains and extended lengths of the multimer. The images we obtained were similar to those reported previously.8 Figure 2A shows more extended chains and more clearly defined globular domains than we were able to obtain previously. Analysis of our images showed vWF molecules over 1 pm in length, but molecular sizes in the range of 200-400 nm were most common. These images confirm that there is considerable variation in size and shape of the vWF macromoleculeswhich results from polydispersity and chain folding. Analysis of AFM images obtained indicated protein height measurements of 3-17 nm (1-6
vWF molecules high). The extended chains displayed features in the 3-6 nm range, while coiled and aggregated chains resulted in the higher features. In control experiments using buffer only or when vWF was substituted by other globular proteins (fibrinogen,plasminogen activator inhibitor, bovine serum albumin) these features, attributed to extended chain molecules, were not observed. The buffer-only solutions produced no features more than 1 nm high. Figure 2B shows a height histogram of Figure 2A. The heights are referenced to the lowest point in the image. The color table used for the inset was chosen so that each change in color corresponds to a 3-nm change in height. The mica substrate appears green, the majority. of the vWF is seen as extended chains (light blue). Aggregates
Eppell et al.
2284 Langmuir, Vol. 9, No. 9, 1993 Table I. Dimensions of von Willebrand Factor Globular Domains measured height across domain (nm)
A B
C D E F
G
H I J
3.5 2.7 3.4 5.8 3.7 3.6 3.0 3.6 5.6 5.6
calculated width width across across (nm) (nm)
91 90 104 127 111 102 89 114 189 127
75 75 88 107 94 86 74 98 169 108
measu r ed height along (nm)
width along (nm)
calculated width along (nm)
3.5 2.5 0.86 2.3 1.4 1.6 0.94 3.8 5.8 3.4
94 99 55 103 125 65 70 128 166 128
78 85 46 89 114 53 61 111 146 112
0 The domains here correspond to the globular units identified in Figure 2D. The measured heights and widths were determined using the same procedure described in Figure 3B. The "across" measurements were taken using a line scan that was perpendicular to a line connecting the center of the domain of interest to an adjacent domain. The "along" measurements were taken using a line scan that was parallel to the connecting line. The calculated widths were determined assuming aspherical tip of radius 14 nm and vWF GG domains modeled as rectangles with circular end caps.
and coiled chains appear as dark blue and magenta. It is apparent that the taller regions appear to be aggregates and coiled portions of the extended chains. The histogram shows two peaks. The sharper peak centered around 2 nm corresponds to the mica substrate. The broader peak centered around 5 nm corresponds to the extended vWF chains. Thus, the extended vWF is approximately 3 nm taller than the mica surface. The relatively rare aggregates and coils correspond to the tail of the histogram stretching off to the right. Figure 2C shows a higher magnification of the portion of the multimer in Figure 2A that is designated with an arrow. It is clear that there are separate globular domains strung out like pearls on a necklace. The domains have elliptical cross sections. The orientations of the major and minor axes of the individual globular domains does not appear to be correlated with the overall orientation of the molecules. Characteristic dimensions of the various domains shown in Figure 2C were measured by analyzing line scans through the center of each domain (see Table I). By use of the same technique, all the domains that show as light blue in Figure 2B were analyzed (101 domains). We found a mean height of 3.4 nm (f0.83 nm), a mean major axis of 121 (f22) nm, and a mean minor axis of 95 (f24 nm). The f values are equal to one standard deviation of the relevant data sets. Based on the distance d in Figure 2D being 160 nm, in good agreement with the size of a vWF protomer reported by others,1°-13we associate the domains labeled E, F, and G with the GG domain, symmetry nodule, GG domain, respectively (see Figure 1). Due to their large heights, we attribute the domains D, I, and J to overlapping globular domains. The globular domains are not rigidly attached to each other as is manifested by the meandering of the molecule on the surface. While our images do not show the fibrillar domains connecting the globular domains, this meandering is consistent with the globular domains being connected by flexible fibrous chains. Our AFM data agree with TEM data on a 100 nm length scale as discussed above. However, there are three aspects of the data on the 10 nm length scale where the two techniques do not agree. First, TEM work suggests that there is a small nodule situated between the larger GG domains. We see three roughly equally sized globular
-
domains. Second, TEM work shows fairly conclusively that the globular domains are connected via fibrillar domains that appear as flexible sticks in the TEM images. Again, we see no evidence for these domains in our images. Third, TEM results indicate that the diameter of the GG domains is -30 nm. Our AFM data indicate an average characteristic width of 100 nm. Assuming the response of the calibration characteristics of our AFM is monotonic with respect to length scale, we can conclude that our instrument was accurate to within 20% when measuring the vWF. Thus, the discrepancy between our data and the reported TEM data is not due to miscalibration of the AFM. While the AFM technique has an intrinsically greater precision than TEM, the accuracy of the AFM is still a matter of debate. The systematic enlargement of objects due to finite tip size is a known a r t i f a ~ t . 'The ~ ~ magnitude ~~ of this effect is dependent on the height of the object measured, the ambient humidity,15dand the size and shape of the AFM tip. As the amount of enlargement to be expected when measuring a vWF molecule was not known, we designed an experiment to obtain this information. Fourteen-nanometer polystyrene spheres were employed as a model structure for the globular domains of vWF. Polystyrene Spheres. We hypothesized that our vWF structure was similar to that seen by TEM but that an instrumental artifact caused the protein's image to be distorted. To test this hypothesis, it was important to investigate the instrument response to a surface like vWF on mica. To accomplish this, we needed a suitable model structure of known dimension to replace the vWF. This model structure would also allow us to measure the size of the AFM tip on a nanometer scale. Previous TEM studies indicated that perhaps each GG domain consists of two 13 nm diameter subdomains. We chose as our model object 14 nm diameter polystyrene spheres because they come well characterized by TEM from the manufacturer; they are available in the size and shape pertinent to our protein studies; and at room temperature, the polystyrene is below its glass transition temperature. Thus, it is incompressible and not expected to deform under the load applied by the AFM. Li and Lindsay imaged polystyrene spheres with the AFM.21 They were primarily interested in studying twodimensional crystallization. Thus, they concentrated on imaging large arrays of spheres. We have extended these studies to spheres -3 times smaller than those used by Li and Lindsay and to the limit of very low surface coverage. Figure 3A shows that we were able to obtain surfaces with isolated spheres dispersed on them. The widths of the spheres were determined from line scans as shown in Figure 3B. The results of this analysis are displayed in a histogram, Figure 4. The mean diameter of the spheres was found to be 39 nm ( i t 3 nm), approximately 3 times the width measured by the manufacturer using TEM. With these data it was possible to model the portion of the tip used to image the vWF. It is known from scanning electron microscopy, SEM, data that the shape of the tip on a micrometer scale is pyramidal. (This particular geometry is obtained because the tip mold is made by etching a pit in the (100) face of a Si wafer using an anisotropic etch which stops at (111)planes. This results in a pyramid with (111)faces. This mold is then filled with Si3N4to form the tip.) Ifwe assume the tip terminates in a single atom, the half-angle of the pyramid necessary to explain our results is 70'. The effective width of the portion of the tip used to image the sphere would be 37
-
-
(21)Li, Y.;Lindsay, S.M. Reu. Sci. Instrum. 1991, 62,2630.
Langmuir, Vol. 9,No. 9, 1993 2285
AFM S t u d y of Globular Proteins
I
Figure 3. (A, left) An image typical of the polystyrene spheres on mica surfaces analyzed. Our deposition procedure resulted in the spheres being well separated on the surface. We found it necessary to vacuum dry the polystyrene on mica surfaces for several hours before imaging. Without this step, we saw only streaks on the surface. These were attributed to loosely bound spheres being pushed across the surface by the AFM tip. (B, right) Enlarged region around a sphere and a cross section taken through the center of the sphere. The resulting line scan was then used to measure the width of the sphere. This was done in both the fast scanning direction and at 90" to the fast scanning direction. Over 300 spheres were examined on two different substrates with three different tips. (Our measurement procedure is more accurate than making the measurement from the topographic mode in which only the intensity of the image is used to determine the boundary of the sphere. The latter technique causes one to underestimate the diameter by 10%. This is expected due to the logarithmic response of the human eye with respect to intensity.)
-
Widths of 14nm Spheres Measured by AFM
Bin Width = 2.0nm
I 5
35
Mean Width = 39nm
30
N = 318
; 25 0
L
5
=
20 15 10
5 0 0
20
40
60
80
100
Sphere Diameter (nm)
Figure 4. Widths of the polystyrene spheres on mica as measured by AFM displayed as a histogram. The image in Figure 3 represents a typical portion of the surfaces used to construct the histogram.
nm. This is larger than would be predicted assuming a pyramidal tip formed by the intersection of four (111) planes in a cubic lattice. In that case, one obtains a halfangle of 35' and an effective width of 7.8 nm. It is unlikely that the pyramid actually extends all the way to the apex of the tip. It is more probable that, a few nanometers from the apex, some smooth shape is present. We assumed a spherical tip and calculated the radius necessary to explain our results to be 14 nm. This is consistent with the values extrapolated from SEM images.22 Applying Polystyrene Sphere Results t o v W F Images. This information was applied to the vWF images to obtain more accurate estimates of the dimensions of the molecule (Figure 2). Performing a subtraction anal(22) Albrecht, T. R.; Akamine, W.; Carver, T. E.; Quate, C. F. J. Vac. Sci. Technol., A 1990,8, 3386.
ogous to that used by Bustamante et al.,15aassuming a spherical tip (r = 14 nm) and using the height of the globular domains as measured by AFM, the tip broadening effect was calculated (see Table I). The ellipses superimposed on the AFM image in Figure 2D indicate where the perimeters of the globular domains actually lie. We performed this calculation for all the domains measured in Figure 2A and found that the vWF globular domains that appear as 121 nm X 95 nm ellipses in the AFM images are actually 106 nm X 81 nm ellipses. The 5 nm central nodule seen by TEM would be imaged as having a width of 23.7 nm. Thus, our images of three large globular domains is, to some extent, explained by the instrumental artifact hypothesis. However, the GG domains should still appear almost twice the size of the central nodule and the tip should descend to the fibrillar domain and image -10 nm of the fiber before ascending once again due to the central nodule. It is possible that the humidity in our laboratory caused the lateral extent of the image of the vWF to be exaggerated. Thundat et have shown that high humidity causes the width of DNA on mica, as measured by AFM, to be increased by as much as a factor of 2. AFM Height Anomaly. 'During the course of our polystyrene sphere experiments, we found that the sphere heights, as measured by AFM, were anomalously low. A histogram of the heights for the same data set described in Figures 3 and 4 is presented in Figure 5A. Notice that all of the sphere heights (14 nm by TEM) are
