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Perceived Substratum Characteristics as a Function of AFM Probe and Imaging Fluid Properties J. B. Morrow,† B. F. Smets,†,‡ and D. Grasso*,§ Environmental Engineering Program and Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, and Picker Engineering Program, Smith College, Northampton, Massachusetts 01063 Received March 10, 2003. In Final Form: May 13, 2003 Probe and imaging fluid contributions to substratum root-mean-square (rms) roughness and feature heights, as measured with atomic force microscopy (AFM), were explored. A unique method to measure the roughness of a singular location ((70 nm) was developed. Probe type [glass (hydrophilic) and octadecyltrichlorosilane (OTS)-coated glass (hydrophobic) colloids and Si3N4 (hydrophilic)] were observed to influence feature heights but not surface rms averaged roughness of the glass substratum. Probe type significantly impacted both rms roughness and feature heights on the OTS-coated substratum. Use of smaller Si3N4 probes (80 nm diameter versus 1 µm diameter colloid probes) in air resulted in the largest surface roughness measurements for both substrata. Changes in perceived OTS-coated substratum roughness as a function of probe type were postulated to derive from disordered organosilane surface layers. Interactions of OTS-coated glass colloid probes with the OTS-coated glass substratum decreased observed feature heights. Roughness of the OTS-coated substratum increased when imaged in organic fluids while individual feature heights decreased with increasing fluid polarity. Finally, surface-averaged rms roughness correlated positively with measured adhesive forces. In sum, what were thought to be intrinsic surface properties (topography and chemistry) can be modified by imaging conditions (probe type and imaging fluid).
Introduction Surface topography has been linked to surface properties such as adhesion, gloss, and biocompatibility.1 Surface roughness, a measure of surface topography, is typically recorded via atomic force microscopy (AFM) when a probe is brought into contact with a substratum that is axially translated relative to the probe.2 Roughness measurements performed in contact mode using Si3N4 probes with air as the imaging fluid are often used to interpret and predict particle/surface interactions under a variety of experimental conditions.3,4 Due to the nature of AFM methodology, contributions of probe/substratum interaction to roughness measurements cannot be eliminated.5 Several studies have characterized the impact of probe size and geometry on roughness measurements and imaging resolution.5-9 Vertical roughness measurements, including root-mean-square (rms) roughness, are the least impacted by probe shape.8 Furthermore, molecular-scale * Corresponding author. Fax 413-585-7001. Phone 413-585-7000. E-mail:
[email protected]. † Environmental Engineering Program, University of Connecticut. ‡ Department of Molecular and Cell Biology, University of Connecticut. § Picker Engineering Program, Smith College. (1) Assender, H.; Bliznyuk, V.; Porfyrakis, K. Science 2002, 297, 973976. (2) Takano, H.; Kenseth, J. R.; Wong, S. S.; O’Brien, J.; Porter, M. D. Chem. Rev. 1999, 99, 2845-2890. (3) Shellenberger, K.; Logan, B. E. Environ. Sci. Technol. 2002, 36, 184-189. (4) Jacquot, C.; Takadoum, J. J. Adhes. Sci. Technol. 2001, 15, 681687. (5) Dongmo, S.; Vautrot, P.; Troyon, M. Appl. Phys. A 1998, 66, S819S823. (6) Mannelquist, A.; Almqvist, N.; Fredriksson, S. Appl. Phys. A 1998, 66, S891-S895. (7) Sedin, D. L.; Rowlen, K. L. Appl. Surf. Sci. 2001, 182, 40-48. (8) Westra, K. L.; Thomson, D. J. J. Vac. Sci. Technol. B 1995, 13, 344-349. (9) Weihs, T. P.; Nawaz, Z.; Jarvis, S. P.; Pethica, J. B. Appl. Phys. Lett. 1990, 59, 3536-3538.
surface roughness has been implicated in explaining discrepancies between experimental observations and traditional colloid stability predictions.10,11 Indeed, asperities significantly smaller than the radius of interacting particles have been reported to significantly decrease adhesion to surfaces.12-15 However, it has not yet been reported how probes, interposed imaging fluids, and probe/ substratum interactions contribute to inferred substratum descriptors such as roughness and specific feature resolution. On the basis of reported changes in adhesive interactions,4,16-21 it was hypothesized that imaging fluid polarity and probe characteristics could alter observed surface roughness as measured in contact mode AFM. Hydrophilic and hydrophobic glass substrata were studied with fluids of varying polarity and with AFM probes of three different surface chemistries (glass, octadecyltrichlorosilane (OTS)-coated glass, and Si3N4). Glass is a commonly studied substratum22-25 and its surface chemistry 16 and roughness 3 are easily altered. Substrata of different hydrophobicity were examined to quantify the (10) Bhattacharjee, S.; Ko, C. H.; Elimelech, M. Langmuir 1998, 14, 3364-3375. (11) Suresh, L.; Walz, J. J. Colloid Interface Sci. 1996, 183, 199213. (12) Rabinovich, Y. I.; Adler, J. J.; Ata, A.; Singh, R. K.; Moudgil, B. M. J. Colloid Interface Sci. 2000, 232, 10-16. (13) Rabinovich, Y. I.; Adler, J. J.; Ata, A.; Singh, R. K.; Moudgil, B. M. J. Colloid Interface Sci. 2000, 232, 17-24. (14) Walz, J. Y. Adv. Colloid Interface Sci. 1998, 74, 119-168. (15) Beach, E. R.; Tormoen, G. W.; Drelich, J.; Han, R. J. Colloid Interface Sci. 2002, 247, 84-99. (16) Freitas, A. M., Sharma, M. M. J. Colloid Interface Sci. 2001, 233, 73-82. (17) Hoh, J. H.; Cleveland, J. P.; Prater, G. B.; Revel, J. P.; Hansma, P. K. J. Am. Chem. Soc. 1992, 114, 4917-4918. (18) Weisenhorn, A. L., Maivald, P., Butt, H.-J., Hansma, P. K. Phys. Rev. B 1992, 45, 11226-11232. (19) Vezenov, D. V.; Zhuk, A. V.; Whitesides, G. M.; Lieber, C. M. J. Am. Chem. Soc. 2002, 124, 10578-10588. (20) Abu-Lail, N. I.; Camesano, T. A. Langmuir 2002, 18, 40714081. (21) Kokkoli, E.; Zukoski, C. F. Langmuir 1998, 14, 1189-1195.
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effect of surface character on overall interaction energy. Subsequent chemisorption of a self-assembled monolayer of OTS on etched glass rendered it hydrophobic. The effects of probe and substratum chemical symmetry on substratum descriptors were explored. Using AFM in contact mode, two substratum descriptors (rms roughness and heights of specific features) were quantified for different probe/fluid/substratum combinations. Background When two surfaces come in contact, as in contact-mode imaging, an equilibrium is established that derives from the interaction of the contacting surfaces.26,27 The free energy of adhesion between the probe and the substratum can be described as a function of apolar (van der Waals) and polar (Lewis acid-base) free energies:28
∆G ) ∆GLW + ∆GAB
(1)
The net free energy of adhesion is related to surface tension components, γ, of the respective surfaces. In a vacuum, the net free energy of adhesion accounting for Liftshitzvan der Waals, γLW, and acid-base surface tension components is28
∆Gij ) -2( xγiLWγjLW + xγi+γj- + xγi-γj +)
(2)
where γ+ and γ- are electron-acceptor and electron-donor surface tension components, respectively. The Liftshitzvan der Waals component subsumes Keesom, Debye, and London interactions.28 The total interaction energy can be expanded to include imaging fluids, γk.28
∆Gikj ) -2((xγiLW - xγkLW)(xγjLW - xγkLW) +
(xγi+ - xγj+)(xγi- - xγj-) - (xγi+ - xγk+)(xγi- -
xγk-) - (xγj+ - xγk+)(xγj- - xγk-))
(3)
The force of adhesion, Fad (i.e., the requisite force to detach a probe from the surface),17 is directly related to the overall interaction energy and can be compared to the experimentally determined single-site force of adhesion as measured by AFM.29 The force can be predicted from the net free energy of interaction, ∆G, between two surfaces (a spherical probe of radius R and a flat plate substratum) in a suspending liquid according to the Derjaguin approximation,26
F ) -2πR∆G
(4)
Deviations of measured forces from predicted interactions have been attributed to a variety of parameters, including surface roughness, steric, hydration, hydrophobic, capillary, and friction forces30-32 (see Grasso et (22) Bergendahl, J.; Grasso, D. AIChE J. 1999, 45, 475-484. (23) Das, S. K.; Sharma, M. M.; Schechter, R. S. Particulate Sci. Technol. 1995, 13, 227-247. (24) Grasso, D.; Smets, B. F.; Strevett, K. A.; Machinist, B. D.; van Oss, C. J.; Giese, R. F.; Wu, W. Environ. Sci. Technol. 1996, 30, 36043608. (25) Rijnaarts, H. H. M.; Norde, W.; Bouwer, E. J.; Lyklema, J.; Zehnder, A. J. B. Environ. Sci. Technol. 1996, 30, 2877-2883. (26) van Oss, C. J. Interfacial Forces in Aqueous Media; Marcel Dekker: New York, New York, 1994. (27) Israelachvili, J. Intermolecular and Surface Forces, 2nd ed.; Academic Press Inc.: San Diego, CA, 1992. (28) van Oss, C. J.; Chaudhury, M. K.; Good, R. J. Chem. Rev. 1988, 88, 927-941. (29) Kappl, M.; Butt, H. J. Part. Part. Syst. Charact. 2002, 19, 129143.
al.30 for a comprehensive review of non-DLVO interactions). Although qualitative agreement between measured forces of adhesion of hydrophobic surfaces in aqueous media and predicted interaction energies have been reported, hydrophobic forces can contribute to deviations of observation from traditional theoretical predictions.16,33 Environmental conditions can also significantly influence measured adhesive forces. For instance, increasing the ionic strength of the fluid can reduce monotonic hydration forces.34 Others have noted quantifiable differences from predictions in the measured adhesive force as a result of changes in imaging fluid polarity,4,18-20 ionic strength,21 and pH.17 Moreover, conducting measurements in fluids has been reported to eliminate capillary forces31 and reduce friction forces31 commonly observed in air. Our work sought to explore whether a relationship exists between AFM probe/substratum interactions in fluids of varied polarity and subsequent inferred surface descriptors. Methods Reagents. All reagents used were analytical grade except for diiodomethane, which was laboratory grade (Fisher Scientific, Pittsburgh, PA), unless otherwise noted. Prior to imaging, all fluids were filtered through 0.2 µm PTFE membrane filters (Nalgene Nunc International Corp. Rochester, NY). Substrata Preparation. Glass surfaces were cut to approximately 1 cm by 1 cm squares from soda lime microscope slides (Fisherbrand, Fisher Scientific, Pittsburgh, PA). Glass substrata were cleaned by soaking successively for 24 h in 37.4% HCl, 10% H2CrO4, and 37.4% HCl. Substrata were rinsed with deionized water between acid bath washes.3 Acid etching was used to increase the glass surface roughness.3 After acid treatment, glass substrata were rinsed in deionized water (Milli-Q, Millipore Corp., Bedford, MA resistivity 18 MΩ), allowed to airdry, and stored in a desiccator. Immediately prior to imaging, hydrophilic glass substrata were exposed to 15% HNO3 for 1 h, rinsed with filtered deionized water and filtered ethanol (99%) (Pharmco, Brookfield, CT), and dried with triple-filtered air (once through 0.8 µm and twice through 0.2 µm PTFE membrane filters). Hydrophobic glass substrata were prepared by chemisorption of a self-assembled monolayer of OTS on cleaned glass slides per established protocols.16 Once the glass was cleaned, it was soaked for 30 min in a 15% HNO3 solution to eliminate organic contaminants and activate the surface silanol groups, rinsed with filtered deionized water, and then dried with triple-filtered air. The glass was completely submerged for 16 h in a 1% (w/w) solution of OTS (Aldrich Chemical Co. Inc.) in toluene. Glass slides were removed and rinsed in toluene to remove excess OTS and allowed to air-dry. Immediately after the OTS-modified substratum was imaged in air with the OTS probe, it was reimaged with a Si3N4 probe, yielding the same topography previously recorded with a Si3N4 probe, confirming that the OTScoated glass colloid probe did not physically disrupt the OTScoated substratum. AFM Measurements. All surface roughness measurements were performed with a Digital Instruments Nanoscope IIIa controller and Multimode SPM scanner (Digital Instruments, Santa Barbara, CA). The same location on the glass samples was located ((70 nm) using a small indentation placed in the center of the steel puck prior to mounting the glass sample. Once the center of the indentation in the steel puck was located and centered, the tip of the probe was brought into focus and aligned to the same location for all subsequent measurements. (30) Grasso, D.; Subramaniam, K.; Butkus, M.; Strevett, K.; Bergendahl, J. Re/Views Environ. Sci. Bio/Technol. 2002, 1, 17-38. (31) Feiler, A.; Larson, I.; Jenkins, P.; Attard, P. Langmuir 2000, 16, 10269-10277. (32) Weisenhorn, A. L.; Hansma, P. K.; Albrecht, T. R.; Quate, C. F. Appl. Phys. Lett. 1989, 54, 2651-2653. (33) Yoon, R. H.; Flinn, D. H.; Rabinovich, Y. I. J. Colloid Interface Sci. 1997, 185, 363-370. (34) Butt, H. J.; Jaschke, M.; Ducker, W. Bioelectrochem. Bioenerg. 1995, 38, 191-201.
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The centered probe was engaged with the substratum and a 50 µm × 50 µm section imaged at a scan rate of 1.0 Hz. Surface features observed in air were used to confirm the location when imaging in various fluids. Sections of the height images (50 µm × 50 µm) obtained in contact mode with different substratum, fluid, and probe combinations were analyzed to determine impacts of imaging fluids on observed roughness. A 10 µm × 10 µm section of the glass image and a 25 µm × 25 µm section of the OTS-coated glass image, which contained several large features, were analyzed for rms surface roughness. Heights of two features on each surface were determined using particle size analysis software. All roughness measurements were performed at room temperature with an acoustic hood placed over the head and scanner. The deflection setpoint was established to maintain a constant deflection of 1 V ((0.2 V). Cantilever deflections were converted to corresponding forces (F ) kd, where k is the spring constant of the cantilever and d is the cantilever deflection). Instrument sensitivity was 11 nm/V. An imaging force of 1.13 nN was used for OTS and glass substrata resolution.9 This force corresponds to an average imagining pressure of 0.06 MPa exerted on the substrata during imaging with the Si3N4 probes:35,36
P h)
[ ]
1 16FE2 π 9R2
1/3
(5)
where E is the reduced Young’s modulus for Si3N4 probe/OTS substratum interaction, according to 1/E ) (1 - ν12)/E1 + (1 - ν22)/E2, and νi are the Poisson ratios for a sphere and flat plate. Ei and νi values for Si3N4 were 2.8 × 108 N/m and 0.2, respectively, and for the OTS, 9.3 × 109 N/m and 0.4, respectively.9,37 Garcia-Parajo et al.36 reported that 5.4 GPa was required to compress OTS polymers on silica surfaces with silica probes (10 nm diameter). However, when we used their data to recalculate this required pressure, we found it to be 5.4 MPa. In either case, our imaging pressure was much lower than either of these values. Garcia-Parajo et al.36 also reported a spring constant of 1 N/m for OTS polymers. This is significantly larger than the spring constants of cantilevers used in our work. We measured cantilever spring constants of 0.103 ( 0.001, 0.250 ( 0.001, and 0.249 ( 0.001 N/m for Si3N4, glass, and OTS-coated glass colloid probes, respectively. Applying cantilevers with spring constants significantly smaller than the spring constant of the imaged substrata enabled collection of accurate roughness measurements. Force curves were taken at 15 locations in a 10 µm radius from probe centered positions in a 50 µm × 50 µm area for each liquid. Identifying the same location ((70 nm) allowed for comparison of measurements among different liquids. Force curves were collected in tapping mode by setting the drive amplitude to zero, stopping cantilever rastering, and zeroing as described elsewhere.38 In some cases, cantilever deflection exceeded the split in the photodiode and appeared to bottom-out while adhesive force curves were collected. This has been attributed to low cantilever spring constants.34 In these cases (marked with asterisks in Table 3), the maximum adhesive force, just prior to disjoining, was recorded. The actual adhesive force is at least as large as those expressed in Table 3. Force curves collected in air in contact mode were acquired approximately halfway between the identified features on each glass substrata. To eliminate laser reflection interference, force curve measurements in air were collected with a low coherence length laser (Digital Instruments Nanoscope IIIa controller and Multimode SPM scanner with Picoforce head). Cantilevers with probes of different surface chemistries were used to quantify roughness and force interactions. Digital Instruments Si3N4 cantilevers (model DNP) were soaked in 15% nitric acid for 15 min and rinsed in filtered deionized water and (35) Du, Q.; Xiao, X.; Charych, D.; Wolf, F.; Frantz, P.; Shen, Y. R.; Salmeron, M. Phys. Rev. B 1995, 51, 7456-7463. (36) Garcia-Parajo, M.; Longo, C.; Servat, J.; Gorostiza, P.; Sanz, F. Langmuir 1997, 13, 2333-2339. (37) Kaye, G. W. C. Tables of Physical and Chemical Constants, and Some Mathematical Functions, 13th ed.; Wiley: New York, 1966. (38) Camesano, T. A.; Logan, B. E. Environ. Sci. Technol. 2000, 34, 3354-3362.
Table 1. Substrata Contact Angle Measurements contact angles (deg), n ) 9 substratum
water
formamide
diiodomethane
glass OTS glass
9.73 ( 1.37 102.65 ( 1.73
9.73 ( 1.56 97.37 ( 1.93
43.01 ( 3.87 70.92 ( 1.62
Table 2. Calculated Surface Free Energy Parameters
surface glassa OTS glassa Si3N4b
surface tension components (mJ/m2) γSLW γS+ γS38.31 16.71 38.59
1.61 0.07 4
53.61 0.77 33.98
Hamaker constant,c Aii (J) 7.21 × 10-20 3.14 × 10-20 7.26 × 10-20
a Calculated according to eq 6. b Reported previously by Freitas and Sharma.16 c Calculated, Aii ) 24πdo2γSLW, where do ) 0.158 nm.26
filtered ethanol (99%) prior to imaging. Cantilevers with 1 µm diameter mounted borosilicate glass colloids were acquired from Novascan Technologies (Iowa State University Research, Ames, IA). Hydrophilic glass colloid cantilevers were pretreated by rinsing for 30 s in 15% HNO3, filtered deionized water, and filtered ethanol (99%) prior to use. OTS-coated colloid cantilevers were prepared from glass colloids as described above by soaking in a 1% OTS solution and rinsing in toluene. Hydrophobic colloid cantilevers were rinsed in deionized water and ethanol prior to use. Contact Angle Measurements. Lewis acid-base interaction energies were determined as described by van Oss.26 Contact angles were acquired for three probe liquids: deionized water (polar), formamide (polar), and diiodomethane (apolar) at 26 ( 1 °C using a Rame´-Hart Goniometer (Model 100-00, Mountain Lake, NJ). Nine separate measurements with each liquid (2 µL) were obtained (Table 1). Contact angle measurements (θis) obtained with the three liquids, were used to solve the YoungDupre´ equation.28
γi(1 + cos θis) ) 2(xγiLWγsLW + xγi+ γs- + xγi- γs+) (6) Net Free Energy Predictions. Net free energies of interaction were predicted by applying the continuum model developed by van Oss-Chaudhury-Good (VCG)27 and surface free energies (Table 2). The VCG model combines van der Waals (∆GLW) and acid-base (∆GAB) free energies to predict the net free energy of interaction at the distance of closest approach (eqs 2 and 3). Net free energies were converted to forces using eq 4. Statistical Comparison. All pairwise comparisons employed t-statistics at R ) 0.05. The p-values for falsely rejecting the null hypothesis of no difference in compared means are also reported.
Results Probe Effect on Substratum Characteristics. Effects of probe type on specific feature heights and a surfaceaveraged descriptor (rms roughness) of substrata were quantified (Figures 1 and 2). To reduce the contribution of optical interference,39 rms roughness was calculated on a portion (dashed line in Figures 1 and 2) of the original 50 × 50 µm image. Within the reduced area, two features of interest were identified (circles in Figures 1 and 2). Surface images along a line between the two features were isolated (panels A-F in Figures 1 and 2). Images were collected in air with Si3N4 and OTS-coated and uncoated glass colloid probes (Panels A-C of Figures 1 and 2), and heights of features were determined. Results of both feature height and surface roughness determinations are discussed according to substratum hydrophobicity and imaging conditions. (39) Mendez-Vilas, A.; Gonzalez-Martin, M. L.; Nuevo, M. J. Ultramicroscopy 2002, 92, 243-250.
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Table 3. Summary of Imaging and Interaction Data for Various Substratum/Fluid/Probe Combinations
probe type
rms roughness (nm)
height G1 (nm)
height G2 (nm)
air (1.00054) diiod (5.3) water (80) form. (109.5) air (1.00054) diiod (5.3) water (80) form. (109.5) air (1.00054) diiod (5.3) water (80) form. (109.5)
0.27 ( 0.01 0.23 ( 0.02 0.22 ( 0.02 0.25 ( 0.05 0.29 ( 0.02
2.15 ( 0.14 1.74 ( 0.51 1.05 ( 0.13 0.83 ( 0.23 1.12 ( 0.10
1.64 ( 0.17 1.73 ( 0.09 1.39 ( 0.10 1.06 ( 0.14 2.09 ( 0.38
0.25 ( 0.04
1.85 ( 0.29
2.12 ( 0.28
substratum
Si3N4a
glass
glassb
glass
OTS glassb
glass
probe type
fluid (dielectric constant)c
substratum
Si3N4a
OTS-coated glass
glassb
OTS-coated glass
OTS glassb
OTS-coated glass
fluid (dielectric constant)c
rms roughness (nm)
height O1 (nm)
height O2 (nm)
air (1.00054) diiod (5.3) water (80) form. (109.5) air (1.00054) diiod (5.3) water (80) form. (109.5) air (1.00054) diiod (5.3) water (80) form. (109.5)
0.71 ( 0.03 0.53 ( 0.05 0.48 ( 0.04 0.62 ( 0.04 0.48 ( 0.01
11.6 ( 4.98 12.77 ( 0.93 10.08 ( 0.72 9.84 ( 1.11 13.52 ( 1.18
23.01 ( 2.40 21.34 ( 1.44 18.47 ( 0.26 16.09 ( 1.80 19.19 ( 1.21
0.47 ( 0.01
2.45 ( 0.31
1.86 ( 0.14
measured adhesive forced and predicted adhesive forcee (mN/m) -1267.4, -1313.8, 662.5 -111.6 ( 7.2, -171.7 -471.8 ( 214.8,f 270.3 -97.6 ( 8.3, 14.4 -175.9, -137.1, 715.1 -18.5 ( 7.6, -222.6 -28.8 ( 4.1, 244.9 -15.6 ( 9.1, 6.2 -222.4,f -271.9,f 355.7 -0.7 ( 0.2, -2.0 -26.7 ( 8.8,f -72.4 -15.5 ( 8.9, 1.4 measured adhesive forced and predicted adhesive forcee (mN/m) -646.5, -435.6 (O2), 348.6 -13.1 ( 4.0, 4.8 -55.4 ( 15.7, -91.1 -19.0 ( 8.6, -34.7 -210.3,f -520.7f (O2), 355.7 -27.6 ( 14.6, -2.01 -24.1 ( 1.9,f -72.4 -24.8 ( 10.9, 1.4 -174.9, -169.6 (O2), 215.6 -28.6 ( 20.0, 110.4 24.1 ( 11.3,f -497.9 -50.3 ( 3.1,f -111.7
a Standard deviations reported for Si N probes are for five measurements. b Standard deviations reported for colloid probes are for three 3 4 measurements. c van Oss.26 d Indicates force measured on background substratum at 15 different locations in liquids; two different locations in air both on background substratum for the glass sample and one at background and one at the apex of feature O2. A negative value indicates adhesion, normalized for probe radius. e Calculated on the basis of the van Oss-Chaudhury-Good 27 continuum model; negative values indicate adhesion, and values are normalized for probe radius. f Cantilever deflection exceeded split in photodiode.
Glass Substratum. Rms roughness values for a 10 µm × 10 µm section of glass (Figure 1) as a function of probe chemistry was calculated (Table 3). There was no significant difference in inferred surface roughness among probe types (R ) 0.05). To explore the impact of probe surface chemistry on substratum appearance, heights of two identified features were analyzed (Figure 1, panels A-C). Feature G1 was significantly larger (R ) 0.05) when imaged with Si3N4 (p ) 0.001) and OTS-coated glass colloid (p ) 0.018) probes than when imaged with glass colloid probes. Feature G2 was significantly larger when imaged with OTS-coated colloid probes (p ) 0.015). However, no significant difference in height of G2 was observed using either Si3N4 or glass colloid probes. OTS-Coated Glass Substratum. A 25 µm × 25 µm section of OTS glass (Figure 2) was used to determine effects of probe chemistry on calculated rms roughness (Table 3). A larger area was required to isolate two significant features on the substratum surface. Roughness was significantly larger (R ) 0.05) when imaged with Si3N4 as compared with glass colloid (p ) 0.001) and OTS colloid (p ) 0.002) probes. Again, there was no significant difference in surface roughness between the two colloid probes (p ) 0.18). Probe type had a significant impact on observed feature heights for the OTS-treated substratum (Figure 2, panels A-C). When the OTS substratum was imaged with an OTS-coated glass colloid probe (Figure 2, panel B), the observed heights of both features decreased significantly (R ) 0.05) compared to imaging with hydrophilic probes (glass colloid, panel A, or Si3N4, panel C). Features O1 and O2 were observed to be significantly larger (R ) 0.05) with the hydrophilic [glass colloid (p1 ) 0.002 and p2 )
0.001) and Si3N4 (p1 ) 0.03 and p2 ) 0.003)] probes than with the OTS-coated colloid probe. The two troughs adjacent to O1 and after O2 when imaged with colloid probes are artifacts of tip geometry.40 Effect of Imaging Fluid Polarity on Substratum Characteristics. Contact mode images of the same (50 µm × 50 µm) areas were also captured under fluids of increasing polarity using only Si3N4 probes. Resulting surface roughness and observed feature heights were also quantified (Table 3). Glass Substratum. The glass substratum was significantly rougher (R ) 0.05) when imaged in air than when imaged in water (p ) 0.012) or diiodomethane (p ) 0.003) but not when imaged with formamide (Table 3). Feature heights diminished with increasing imaging fluid polarity (panels C-F of Figure 1). G1 was observed to be significantly larger (R ) 0.05) in diiodomethane than in water (p ) 0.01) or formamide (p ) 0.02). G1 was also significantly larger in air than in water (p ) 0.0001) and formamide (p ) 0.0006). The same trend was apparent for G2, which was significantly larger (R ) 0.05) in diiodomethane than in water (p ) 0.002) and formamide (p ) 0.0002). Furthermore, G2 was significantly larger (R ) 0.05) in air than in both water (p ) 0.003) and formamide (p ) 0.0003). Decreases in feature heights strongly correlated with fluid polarity increases (panel A of Figure 3). OTS-Coated Glass Substratum. Rms roughness decreased with imaging fluids in the following order: air, formamide, diiodomethane, and water (Table 3). Significant differences (R ) 0.05) were measured between air, diiodomethane (p ) 0.003), water (p ) 0.0003), and (40) Xu, S.; Arnsdorf, M. F. J. Microsc. 1994, 173, 199-210.
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Figure 1. Glass substratum contact mode image and section analysis. A 50 µm × 50 µm 3-D image of a glass substratum imaged in air with Si3N4 probe. Section analysis: (A) glass colloid probe in air, (B) OTS coated colloid probe in air, (C) Si3N4 probe, air, (D) Si3N4 probe, diiodomethane, (E) Si3N4 probe, water, (F) Si3N4 probe, formamide. Height bar ) 5 nm. Features G1 and G2 are shown on the 3-D image and section views, and the dotted line indicates the area (10 µm × 10 µm) analyzed for roughness.
formamide (p ) 0.009). Feature O2 decreased with an increase in imaging fluid dielectric constant similar to what was observed for G2 on the glass surface (Figure 2, panels D-F). O2 was observed to be significantly larger (R ) 0.05) in air than in water (p ) 0.004) or formamide (p ) 0.001). O2 was also significantly larger (R ) 0.05) in diiodomethane than in water (p ) 0.006) or formamide (p ) 0.007). Similar trends were not observed for O1, which exhibited similar feature heights for polar liquids (water and formamide) and air, although the height was significantly larger when imaged in diiodomethane than
when imaged in water (p ) 0.01) or formamide (p ) 0.007). Again, decreased feature height with increased fluid polarity was apparent (panel B, Figure 3). Imaging Fluid Polarity Effect on Probe/Substratum Interaction. Because images are a result of contact between the probe and the substratum, probe/substratum interactions as well as intrinsic substratum topography may affect resulting images. Unfortunately, we were unable to collect roughness and feature height measurements with the colloid probes (in the liquids) due to the mass of the colloid coupled with increased damping of the
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Figure 2. OTS-coated glass substratum contact mode image and section analysis. A 50 µm × 50 µm 3-D image of an OTS coated glass substrate imaged in air with Si3N4 probe. Section analysis: (A) glass colloid probe in air, (B) OTS-coated colloid probe in air, (C) Si3N4 probe, air, (D) Si3N4 probe, diiodomethane, (E) Si3N4 probe, water, (F) Si3N4 probe, formamide. Height bar ) 25 nm. Features O1 and O2 are shown on the 3-D image and section views, and the dotted line indicates the area (25 µm × 25 µm) analyzed for roughness.
fluids. This combination caused the cantilever to deflect past the setpoint, initializing a false engage in contact mode. Adhesive forces are a function of the surface free energies of the constituent components, the probe, the substratum, and the intervening imaging fluid properties.27 The force of adhesion, Fad, at two different locations on both glass and OTS glass substrata with Si3N4 in air were measured to be 1290 ( 33.0 and 852 ( 291 mN/m, respectively. Fad was then measured at 15 nearly identical ((70 nm) locations on each substratum with the different probe types in the various fluids. Imaging fluids appeared to impact interaction as measured by Fad between the probe and substratum for several combinations (Figure 4).
Large interaction forces were measured between Si3N4 probes and the glass substratum in all three liquids. Measured forces of adhesion decreased in organic liquids compared to those collected in water in virtually all cases. Furthermore, the force of adhesion between OTS-coated colloid probes and the glass substratum was significantly reduced with diiodomethane as the imaging fluid. Imaging fluids did not significantly influence adhesive forces for glass colloid probes on either substratum. Interactions between the Si3N4 probe and OTS-coated substratum were similarly affected by imaging fluid, as noted for the glass substratum. However, Si3N4 probes did not yield substantially larger forces of adhesion than the colloid probes. Furthermore, adhesive forces for
Probe and Fluid Impacts on Substratum Imaging
Figure 3. Impact of fluid polarity on substrate feature height. Feature height obtained with Si3N4 probe in fluids of increasing dielectric constant (): 9, air ( ) 1.00054); 2, diiodomethane ( ) 5.3); [, water ( ) 80); b, formamide ( ) 109.5); black symbols for glass substratum, gray symbols for OTS-coated glass substratum, filled symbols indicate G1, O1, and open symbols indicate G2, O2. The black lines are based on linear regression analyses; dotted lines are 95% confidence limits.
Figure 4. Impact of fluid polarity on probe/substrate interaction. Measurements were taken at the same 15 locations ((70 nm) on both glass and OTS-coated glass substrata in fluids of increasing polarity: black, diiodomethane ( ) 5.3); dark gray, water ( ) 80); light gray, formamide ( ) 109.5). Adhesive forces (normalized for probe radius) were measured with three different probes, including Si3N4, glass colloid, and OTS-coated colloid probes. Error bars represent standard deviation, n )15.
all probes on the OTS-coated substratum were not significantly reduced with diiodomethane as the imaging fluid. Discussion Perceived Substratum Characteristics. Effects of probe and substratum chemical composition similarity
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on resulting substratum descriptors in air were explored. Glass substratum rms roughness was not affected by probe character, as glass and OTS-coated colloid probes resulted in the similar roughness values. However, probe chemistry did influence the height of glass substrata features. Glass feature, G1, was larger when imaged with an OTS-coated colloid probe (chemical asymmetry) than a glass colloid probe (chemical symmetry). Probe chemistry had no effect on the height of feature G2. Similarily, when the OTScoated substratum was imaged with both glass and OTScoated glass colloid probes, rms roughness values were not significantly different. Furthermore, as was observed for the glass substratum, when an asymmetric imaging system involving a glass colloid probe and an OTS-coated substratum in air was employed, feature heights increased. O1 and O2 were both significantly larger when imaged with the glass colloid probes than OTS-coated colloid probes. Individual feature heights (except for G2) and rms roughness values were the same or larger for both substrata when imaged with a Si3N4 probe in air, a common imaging scenario.3,4 This may be attributed to probe geometry. Glass colloid probes (1 µm approximately diameter) were significantly larger than the Si3N4 probes (80 nm approximately diameter). Although diameters of the OTS-coated probes were not independently confirmed, any contribution of the OTS layer to probe size is most likely insignificant, as the resolution of surface features on the glass substratum were similar to both OTS-coated colloid and glass colloid probes (panels A and B of Figure 1). It has been reported that an increase in tip size from 15 to 40 nm can result in decreased measured substratum roughness. A narrow tip can more accurately probe the troughs between features.7 Surprisingly, the significant reduction in resolution of the OTS-coated substratum features observed with the OTS-coated colloid probe, as compared with Si3N4 or the glass colloid probes, did not translate to a concomitant reduction in measured substratum roughness. Changes in individual feature heights observed with OTS-coated colloid probes in organic fluids can therefore be attributed to feature chemistry rather than probe geometry. Moreover, rms roughness values in air inferred for both substrata were the same, employing either glass colloid or OTS-coated colloid probes. Force curves collected at both the apex of feature O2 and the background substratum revealed a stronger interaction between the OTS-coated colloid probe and the OTS-coated substratum at the apex (Figure 5). Approach curves collected with a glass colloid probe indicated a slightly larger repulsion at the apex of O2 over the background. The best-fit Hamaker constant (2 × 10-20 J) at the distance of closest approach for the OTS/OTS interaction is comparable to a previously reported value of 1 × 10-20 J.4,41 The small repulsion of the glass colloid probe compared to an increased attraction of the OTScoated colloid probe on feature O2 suggests that the OTS probe may be able to track the substratum more closely at the apex of the feature. Imaging Fluid/Substratum Interactions. A complete set of rms roughness and feature height data for uncoated and OTS-coated glass substrata was collected with Si3N4 probes with various imaging fluids. The largest values of roughness and individual feature heights were recorded in air for both substrata. However, the OTScoated glass substratum roughness was significantly higher when imaged in organic fluids than when imaged in water, which may be attributed to increased interaction (41) Parker, J. L.; Claesson, P. M. Langmuir 1994, 10, 635-639.
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Figure 5. OTS-coated glass substratum/colloid probe approach force curves in air. Triangles ) glass colloid probe, diamonds ) OTS-derivatized glass colloid probe. Open symbols indicate the apex of O2, and filled symbols indicate substratum surface measurement. The solid line is the van der Waals fit curve for an OTS-coated glass sphere approaching an OTS flat plane APFS ) 2 × 10-20 J, and the dotted line is the van der Waals model (F/R ) APFS/6d2) for a glass sphere approaching an OTS flat plane. APFS ) 1 × 10-21 J26, and the arrows indicate jump to contact.
of the organic fluids with a disordered layer of organosilane on the glass substratum. Diiodomethane can interact with the methylene groups of the OTS, yielding smaller contact angles on OTS-coated glass than on glass substrata. Nonuniform or disordered layers of OTS may result in exposed silanol groups and larger contact angles. This is consistent with the 70.92° ( 1.62° measured here as compared with 61° reported elsewhere for similar reaction conditions.42 A nonuniform layer of OTS may have significantly contributed to the OTS substratum roughness (OTS-coated glass substratum rms, 0.71 ( 0.03 nm compared to glass substratum, 0.27 ( 0.01 nm in air) by altering substratum topography. Increased interaction of the organic fluids with the organosilane might further alter the conformation and the subsequent perceived roughness. Modeling Probe/Substratum Interactions. As expected, the measured force of adhesion between the AFM probe and substratum was dependent on both the polarity of the imaging fluid and probe characteristics. Largest interactions were noted when measurements were made with a Si3N4 probe on either the glass or OTS-coated glass substrata in water, which is a common measurement scenario4,16,18 (Figure 4). Average Si3N4 force measurements in water and formamide (18.9 ( 8.6 and 3.9 ( 0.3 nN, respectively) were similar to those reported elsewhere for similar surfaces in water and formamide.4 Force measurements between the Si3N4 probe and the OTScoated glass substratum correlated well with VCG model predicted values (Figure 6). However, VCG model predictions underestimated force measurements between the Si3N4 probe and the glass substratum in water and formamide. This is consistent with other reports.4 Discrepancies between measured and predicted Fad have been attributed to changes in Si3N4 probe chemistry, from Si3N4 to the hydrophobic SiO2, as a result of chemical reactions with water.4 Although, this may account for a decrease in repulsive interaction energy between Si3N4 and the glass substratum (predicted Fad/R of 270.3 N/m for Si3N4 to 197.1 mN/m for SiO2, as calculated with surface energy components from Jacquot and Takadoum4), it cannot account for the adhesive force disparities measured with colloid probes. When air was the imaging fluid, the surface-averaged free energy of interaction predicted by the VCG model did (42) Fadeev, A.; McCarthy, T. J. Langmuir 2000, 16, 7268-7274.
Figure 6. Predicted versus measured force of adhesion in fluids. Force measurements performed with Si3N4 probe on glass (A) and OTS-coated glass (B) substrata in liquids of increasing polarity ( ) dielectric constant); 2, diiodomethane ( ) 5.3); [, water ( ) 80); b, formamide ( ) 109.5); at same 15 locations (( 70 nm). Regression analysis lines shown.
not accurately predict measured adhesive forces for different probe/substratum combinations in air (adhesion was observed in all cases where VCG model predicted repulsion, Table 3). Capillary forces from adsorbed water molecules, which are known to complicate force measurements, may, in part, explain this discrepancy.43,44 The Laplace pressure 27 contribution deriving from water capillary adhesive forces was estimated to be 905.0 mN/m for the Si3N4 probe used in this work. However, capillary forces can only account for 69-71% of the adhesive forces of Si3N4 in contact with glass in air. Surface-averaged VCG-predicted forces did not correlate with apparent substratum roughness as determined in the various fluids of increasing polarity. However, substratum rms roughness increased with increasing measured adhesive forces (Table 3). Surface-averaged rms roughness values correlated better with measured individual probe/substratum interactions than with predicted surface-averaged continuum interactions. Conclusion This work explored how AFM probes, imaging fluids, and subsequent probe/substratum interactions impact perceived rms roughness and feature resolution. A unique method was developed in order to measure the roughness, feature height, and adhesive forces at the same location ((70 nm) on substrata under various imaging conditions. (43) Maurice, P.; Forsythe, J.; Hersman, L.; Sposito, G. Chem. Geol. 1996, 132, 33-43. (44) Fuji, M.; Machida, K.; Takei, T.; Watanabe, M.; Chikazawa, M. J. Phys. Chem. B 1998, 102, 8782-8787.
Probe and Fluid Impacts on Substratum Imaging
As expected, the measured adhesive forces were dependent on substratum hydrophobicity and imaging fluid polarity. The van Oss-Chaudhury-Good surface-averaged model underpredicted the adhesive interactions measured for the glass substratum imaged in a polar fluid. VCGpredicted interaction energies correlated positively with measured adhesive forces for the OTS-coated substratum. Measured adhesive forces correlated well with rms roughness for both substrata. However, VCG predictions did not correlate with surface-averaged rms roughness measurements for either substratum. Increased interaction of the Si3N4 probe with OTS-coated glass substratum in organic imaging fluids resulted in increased rms roughness measurements and was attributed to polymer conformation. Fluid polarity did not significantly impact rms roughness values for the glass substratum. For both substrata, larger roughness measurements were recorded in air with Si3N4 than with either glass or OTS-coated colloid probes. Differences were attributed to Si3N4 probe geometry. In addition, asymmetric probe/substratum
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chemistries had a significant impact on individual feature heights. Individual feature heights were also dependent on probe type and were found to decrease with increasing fluid polarity. Changes in feature height were attributed to differences between feature and background surface chemistry. This work demonstrated that substratum characteristics, rms roughness, and feature heights can be affected by probe/substratum interaction and the nature of the interposed imaging fluid. Acknowledgment. We are grateful to A. Morrow for the programming assistance and to E. Rufe at Digital Instruments for technical assistance with the AFM and force measurements with the low noise laser. Financial support was provided by way of a U.S. Department of Education Fellowship awarded to J.B.M. and a National Research Initiative Grant from the U.S. Department of Agriculture. LA0344130
