Nanometer-Scale Surface Properties of Mixed Phospholipid

The Journal of Physical Chemistry B 0 (proofing),. Abstract | Full ..... 1. Pressure-Induced Phase Behavior of Single and Mixed Fatty Acids. K. Ekelun...
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Langmuir 1997, 13, 4779-4784

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Nanometer-Scale Surface Properties of Mixed Phospholipid Monolayers and Bilayers Yves F. Dufreˆne,†,‡ William R. Barger,† John-Bruce D. Green,† and Gil U Lee*,† Naval Research Laboratory, Chemistry Division, Code 6177, Washington, D.C. 20375-5342, and Center of Marine Biotechnology, University of Maryland System, Columbus Center, 701 East Pratt Street, Baltimore, Maryland 21202 Received February 27, 1997. In Final Form: May 22, 1997X Mixed distearoylphosphatidylethanolamine (DSPE) and dioleoylphosphatidylethanolamine (DOPE) monolayers and bilayers have been deposited on mica using the Langmuir-Blodgett (LB) technique, as a model system for biomembranes. Investigation with atomic force microscopy revealed phase-separation for both monolayers in air and bilayers in water in the form of microscopic DSPE domains embedded in a DOPE matrix. For the monolayers in air, the step height measured between the higher DSPE phase and the lower DOPE phase was larger than expected from the molecular lengths, and a significant contrast in adhesion and friction was observed despite identical lipid end groups. This unexpected behavior resulted primarily from a difference in the film mechanical properties, the DOPE phase being inelastically deformed by the probe. For the bilayers in water, similar trends were found in terms of height, adhesion, and friction, but an additional short-range repulsive hydration/steric force over the DSPE phase contributed to the observed differences.

Introduction Interactions between cell membranes form the basis of many important biological processes, including molecular recognition, cell adhesion, cell fusion, and intercellular communication. It is well-documented that membrane constituentssphospholipids, glycolipids, and (glyco)proteinssare not always homogeneously dispersed in the membrane but can be organized in lateral microdomains.1,2 These domains reflect the functional specialization of different regions of the membrane and are thought to play an important role in membrane interaction processes. Examples of lateral organization found in cell membranes or simplified model systems are protein channel aggregates in gap junctions,1 glycosphingolipid clusters,2 and phase separation in phospholipid films.3 Supported lipid bilayers on solid substrates prepared with the Langmuir trough are well-defined models for cell surfaces and for investigating molecular events in membranes.4,5 Lipid bilayers with (sub)microscopic lateral organizations can be used to design surfaces patterned with given functionalities. For instance, two-phase lipid films allow one to incorporate a molecule of interest, such as a specific surface receptor,6 into one phase, while the other phase consists of a lipid matrix which serves as a reference surface. Heterogeneous lipid bilayer research is further stimulated by potential applications in biosensor technology7 and in the biofunctionalization of inorganic solids.5 Fluorescence microscopy, Brewster angle mi* Corresponding author: phone, (202) 767-5383; fax: (202) 7673321; e-mail, [email protected]. † Naval Research Laboratory. ‡ Center of Marine Biotechnology. X Abstract published in Advance ACS Abstracts, August 1, 1997. (1) Abney, J. R.; Braun, J.; Owicki., J. C. Biophys. J. 1987, 52, 441. (2) Curatolo, W. Biochim. Biophys. Acta 1987, 906, 111. Rock, P.; Allietta, M.; Young, W. W.; Thompson, T. E.; Tillack, T. W. Biochemistry 1990, 29, 8484. Hakomori, S.-I. Prog. Brain Res. 1994, 101, 241. Palestini, P.; Allietta, M.; Sonnino, S.; Tettamanti, G.; Thompson, T. E.; Tillack, T. W. Biochim. Biophys. Acta 1995, 1235, 221. (3) Helm, C. A.; Mo¨hwald, H.; Kjaer, K.; Als-Nielsen, J. Biophys. J. 1987, 52, 381. Mo¨hwald, H. Thin Solid Films 1988, 159, 1. Slotte, J. P. Biochim. Biophys. Acta 1995, 1238, 118. Schram, V.; Lin, H.-N.; Thompson, T. E. Biophys. J. 1996, 71, 1811. (4) McConnell, H. M.; Watts, T. H.; Weis, R. M.; Brian, A. A. Biochim. Biophys. Acta 1986, 864, 95. (5) Sackmann, E. Science 1996, 271, 43. (6) Egger, M.; Heyn, S. P.; Gaub, H. E. Biophys. J. 1990, 57, 669.

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croscopy, X-ray diffraction, and freeze-etch electron microscopy combined with specific labeling have been used to investigate membrane domain topology, but none of these techniques can provide direct information with molecular spatial resolution. Hence, there is a need for information about the nanometer-scale structure and surface properties of heterogeneous multicomponent biomembranes. Atomic force microscopy (AFM)8 is a surface imaging technique with nanometer-scale lateral resolution and 0.1 Å normal resolution, which operates by measuring the forces acting between a probe and the sample. Many Langmuir-Blodgett (LB) films have been imaged with AFM including phospholipid, fatty acid, and polymerized amphiphilic films.9-12 In particular, the high spatial resolution of AFM has been exploited to investigate the detailed structure of phase-separated LB films.13 AFM has also been used to measure lateral forces (friction),14 (7) Fischer, B.; Heyn, S. P.; Egger, M.; Gaub, H. E. Langmuir 1993, 9, 136. Tamm, L. K.; Bo¨hm, C.; Yang, J.; Shao, Z.; Hwang, J.; Edidin, M.; Betzig, E. Thin Solid Films 1996, 284-285, 813. (8) Binnig, G.; Quate, C. F.; Gerber, Ch. Phys. Rev. Lett. 1986, 56, 930. (9) Marti, O.; Ribi, H. O.; Drake, B.; Albrecht, T. R.; Quate, C. F.; Hansma, P. K. Science 1988, 239, 50. Hansma, H. G.; Gould, S. A. C.; Hansma, P. K.; Gaub, H. E.; Longo, M. L.; Zasadzinski, J. A. N. Langmuir 1991, 7, 1051. Garnaes, J.; Schwartz, D. K.; Viswanathan, R.; Zasadzinski, J. A. N. Nature 1992, 357, 54. Schwartz, D. K.; Garnaes, J.; Viswanathan, R.; Zasadzinski, J. A. N. Science 1992, 257, 508. Zasadzinski, J. A.; Viswanathan, R.; Madsen, L.; Garnaes, J.; Schwartz, D. K. Science 1994, 263, 1726. (10) Zasadzinski, J. A. N.; Helm, C. A.; Longo, M. L.; Weisenhorn, A. L.; Gould, S. A. C.; Hansma, P. K. Biophys. J. 1991, 59, 755. (11) Hui, S. W.; Viswanathan, R.; Zasadzinski, J. A.; Israelachvili, J. N. Biophys. J. 1995, 68, 171. (12) Egger, M.; Ohnesorge, F.; Weisenhorn, A. L.; Heyn, S. P.; Drake, B.; Prater, C. B.; Gould, S. A. C.; Hansma, P. K.; Gaub, H. E. J. Struct. Biol. 1990, 103, 89. Weisenhorn, A. L.; Drake, B.; Prater, C. B.; Gould, S. A. C.; Hansma, P. K.; Ohnesorge, F.; Egger, M.; Heyn, S.-P.; Gaub, H. E. Biophys. J. 1990, 58, 1251. Mou, J.; Yang, J.; Shao, Z. J. Mol. Biol. 1995, 248, 507. (13) Overney, R. M.; Meyer, E.; Frommer, J.; Brodbeck, D.; Lu¨thi, R.; Howald, L.; Gu¨ntherodt, H.-J.; Fujihira, M.; Takano, H.; Gotoh, Y. Nature 1992, 359, 133. Weisenhorn, A. L.; Schmitt, F.-J.; Knoll, W.; Hansma, P. K. Ultramicroscopy 1992, 42-44, 1125. Chi, L. F.; Anders, M.; Fuchs, H.; Johnston, R. R.; Ringsdorf, H. Science 1993, 259, 213. Jacobi, S.; Chi, L. F.; Fuchs, H. J. Vac. Sci. Technol., B 1996, 14 (2), 1503. Solletti, J. M.; Botreau, M.; Sommer, F.; Minh Duc, T.; Celio, M. R. J. Vac. Sci. Technol., B 1996, 14 (2), 1492. Barger, W.; Koleske, D.; Feldman, K.; Kru¨ger, D.; Colton, R. Polym. Prepr. 1996, 37, 606. Colton, R. J.; Barger, W. R.; Baselt, D. R.; Corcoran, S. G.; Koleske, D. D.; Lee, G. U. J. Adhes. Sci. Technol., in press.

This article not subject to U.S. Copyright.

Published 1997 by the American Chemical Society

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mechanical properties, and surface forces15 of materials. The force sensitivity is typically 10-14 N/Hz1/2, allowing one to discern chemical groups16 and to measure individual ligand-receptor interactions.17 AFM is capable of operating in liquid which makes it possible to study biological samples under physiological conditions.18 In this way, high-resolution AFM images of homogeneous hydrated phospholipid bilayers10,11 and supported planar lipidprotein membranes12 have been obtained. Although AFM is a powerful technique, interpretation of the images at a nanometer scale is complicated by the many factors that contribute to their formation, namely, sample surface morphology, mechanical properties, friction, and surface forces. Further, interpretation of force measurements is limited by the fact that the probesample separation is not directly measured. These limitations emphasize the need to develop surfaces exhibiting lateral organization, where the component of interest can be referenced to known properties. In this paper, we demonstrate that two-phase phospholipid monolayers and bilayers can be used to define the probesample separation during AFM imaging and directly correlate the apparent surface topography of the films with their relative mechanical properties and surface forces. The approach developed here has applications for studying the physicochemical properties of cell membranes as well as membrane interaction processes. Materials and Methods Distearoylphosphatidylethanolamine (DSPE) and dioleoylphosphatidylethanolamine (DOPE) were purchased from Matreya, Inc. (Pleasant Gap, PA), and Sigma Chemical Co. (St. Louis, MO), respectively, and their purity (99%) was confirmed by electrospray mass spectrometry. LB films were prepared at 25 °C with a KSV 5000 LB system (KSV Instruments, Helsinki, Finland). The lipids were dissolved at 0.5 mM in chloroform/ methanol (4:1). Pure solutions and equimolar (1:1) mixtures of DSPE and DOPE were used in this study. Monolayers were spread on a triply-distilled water subphase, and after evaporation of the solvent they were compressed at a rate of 1 mN/m per min. All layers were deposited at a constant surface pressure of 25 mN/m, i.e., well below the collapse pressure. Mixed monolayers were made by raising vertically freshly-cleaved mica through the air-water interface. Bilayers were prepared by depositing (upstroke) a first monolayer of pure DSPE on the mica substrates. Subphase water used for the first layer was then discarded and a mixed layer of DSPE/DOPE was spread on a new water subphase. The mixed monolayer was compressed and deposited onto the DSPE-coated mica on the down stroke. The transfer ratios were all 1:1. The resulting bilayers, which are not stable in air, were transferred under water into previously submerged small beakers for transport to the AFM liquid cell. A procedure similar to that described by Zasadzinski et al.10 was then used to mount the bilayer-coated substrates under water in the liquid cell. AFM measurements were made at room temperature (20-25 °C) using an optical lever microscope19 equipped with a liquid cell (Nanoscope III, Digital Instruments, Santa Barbara, CA). Contact mode topographic and friction images were taken in the (14) Mate, C. M.; McClelland, G. M.; Erlandsson, R.; Chiang, S. Phys. Rev. Lett. 1987, 59, 1942. (15) Burnham, N. A.; Colton, R. J. J. Vac. Sci. Technol. 1989, A7, 2906. (16) Burnham, N. A.; Dominguez, D. D.; Mowery, R. L.; Colton, R. J. Phys. Rev. Lett. 1990, 64, 1931. Frisbie, C. D.; Rozsnyai, L. F.; Noy, A.; Wrighton, M. S.; Lieber, C. M. Science 1994, 265, 2071. Green, J.-B. D.; McDermott, M. T.; Porter, M. D.; Siperko, L. M. J. Phys. Chem. 1995, 99, 10960. (17) Lee, G. U; Kidwell, D. A.; Colton, R. J. Langmuir 1994, 10, 354. (18) Drake, B.; Prater, C. B.; Weisenhorn, A. L.; Gould, S. A. C.; Albrecht, T. R.; Quate, C. F.; Cannell, D. S.; Hansma, H. G.; Hansma, P. K. Science 1989, 243, 1586. (19) Meyer, G.; Amer, N. M. Appl. Phys. Lett. 1988, 53, 1045.

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Figure 1. Schematic drawing of the supported LB phospholipid films investigated in this study: (a) mixed DSPE/DOPE monolayer on mica in air; (b) mixed DSPE/DOPE monolayer on DSPE-coated mica in water. constant-deflection mode20 using oxide-sharpened microfabricated Si3N4 cantilevers21 (Park Scientific Instruments, Mountain View, CA) with typical radius of curvature of 20 nm and spring constants of 0.03 and 0.5 N/m (manufacturer specified). The applied force was kept as low as possible (∼1 nN). Scan rates ranging from 0.5 to 10 Hz were tested. The sensitivity of the AFM detector was estimated using the slope of the loading curve. Tapping mode imaging was performed using silicon cantilevers (Nanosensors, Aidlingen, Germany) with a resonance frequency of ∼300 kHz and a spring constant of ∼40 N/m. The setpoint amplitude was held as high as possible to minimize the influence of sample properties on the topographic contrast.22

Results and Discussion Toward the goal of developing a versatile model for heterogeneous biomembranes for AFM studies, mixed LB phospholipid monolayers and bilayers were prepared (Figure 1). Phase separation was obtained by dissolving two immiscible23 phospholipids, DSPE (saturated fatty acyl chains) and DOPE (containing a cis-double bond in both fatty acyl chains), in chloroform/methanol in a 1:1 molar ratio. Monolayers formed from this solution were transferred onto mica (Figure 1a) and DSPE-coated mica (Figure 1b), and the resulting supported monolayers and bilayers were investigated by AFM in air and water, respectively. The surface pressure (π) vs area isotherms (25 °C) of DSPE, DOPE, and mixed DSPE/DOPE (1:1) monolayers at the air-water interface are shown in Figure 2. The shapes of the isotherms indicate that the saturated DSPE monolayer is characterized by a two-dimensional (2-D) solid-like organization, whereas the unsaturated DOPE monolayer has a 2-D liquid-like behavior. The isotherm for the mixed DSPE/DOPE film falls between those of the two pure monolayers. At a deposition pressure of 25 mN/ m, DSPE and DOPE occupy 41 and 67 Å2/molecule, respectively, and their surface compressional moduli24 are found to be 183 ( 5 and 79 ( 1 mN/m, indicating that DSPE is stiffer in the horizontal plane than DOPE. Monolayers in Air. The topographic, friction, and adhesion images of a mixed DSPE/DOPE monolayer on mica in air are presented in Figure 3. Phase separation is clearly observed in all images, the film being made of (20) In the constant-deflection mode, a feedback loop is used to keep the normal deflection of the cantilever constant. The force applied to the surface is held constant in this imaging mode. (21) Albrecht, T. R.; Quate, C. F. J. Vac. Sci. Technol., A 1988, 6, 271. (22) Burnham, N. A.; Behrend, O. P.; Oulevey, F.; Gremaud, G.; Gallo, P.-J.; Gourdon, D.; Dupas, E.; Kulik, A. J.; Pollock, H. M.; Briggs, G. A. D. Nanotechnology, in press. (23) Van Dijck, P. W. M.; De Kruijff, B.; Van Deenen, L. L. M.; De Gier, J.; Demel, R. A. Biochim. Biophys. Acta 1976, 455, 576. (24) Gaines, G. L., Jr. Insoluble monolayers at liquid-gas interfaces; Interscience Publishers: New York, 1966; p 24. (25) Baselt, D. R.; Baldeschwieler, J. D. J. Appl. Phys. 1994, 76, 33.

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Figure 2. Surface pressure vs area per molecule isotherms for DSPE, DOPE and mixed (1:1) DSPE/DOPE monolayers at the air-water interface (25 °C).

well-defined elliptical domains ∼5-10 µm in size, embedded in a continuous matrix. Two pieces of evidence allow one to assign the higher level (elliptical domains) to DSPE and the lower level (matrix) to DOPE. First, a simple space-filling model of the minimum energy configuration of the molecules indicates that the length of DSPE, 3.3 nm, is larger than that of DOPE, 2.9 nm. Second, the area fraction covered by the elliptical domains, 37 ( 5%, is in agreement with the area fraction of DSPE in the mixed monolayer at the air-water interface, 38%, calculated for a 1:1 DSPE:DOPE molar ratio and areas per molecule of 41 Å2 for DSPE and 67 Å2 for DOPE. The step height27 measured between the two phases is 1.3 ( 0.2 nm (Figure 3a), which is 0.9 nm larger than the 0.4 nm height difference expected from the simple spacefilling models. To understand the effect of imaging force on the measured height, the thicknesses of the DSPE and DOPE films were directly measured as a function of applied load by scraping the monolayer away with the AFM probe in a defined area to reveal the mica substrate.28 In Figure 4, the measured thickness (thickness minus depth of penetration) of DSPE and DOPE layers is plotted vs F2/3, where F is the externally applied force. In its simplest form the elastic deformation in the contact area between a sphere pressed into a flat can be described by the Hertz theory,29 in which the depth of penetration increases with the 2/3 power of the force applied on the surface.30 Interestingly, the two lipid films exhibit very different behaviors: while the DSPE layer is gradually compressed with increasing loads, in agreement with an (26) Adhesive force maps were obtained by recording 64 × 64 force curves per image. From this force curve arrays, x-y adhesion maps were generated by displaying the pull-off force measured for each force curve. Images were resampled to 512 × 512 pixels. (27) The height differences were measured from cross sections in the topographic images. At forces ∼1 nN, due to variations in the material properties of the two lipids, the step height varied from one probe to another as a result of variations in the radius of curvature of the probe. We present a mean value and standard deviation of height differences from six different images. (28) The monolayer was cut by imaging 1 µm × 1 µm areas at large forces (>1500 nN) and high rates (60 Hz) for short period of times. Larger images of these areas under normal loads with new probes revealed terraces with 1 nm step heights which is consistent with half the unit cell of the muscovite mica lattice along the c axis (Hu, J.; Xiao, X.-d.; Ogletree, D. F.; Salmeron, M. Surf. Sci. 1995, 327, 358). Complete removal of the lipid film allowed us to unambiguously measure the film thickness. (29) Johnson, K. L. Contact Mechanics; Cambridge University Press: Cambridge, U.K., 1985. (30) In the presence of attractive forces such as observed in this study, the effective force applied on the surface is equivalent to the sum of the externally applied force and of the adhesive pull-off force.

Figure 3. Gray scale images (15 µm × 15 µm) of a mixed DSPE/DOPE monolayer on mica in air: (a) topography (zrange: 20 nm), (b) friction, and (c) adhesive force.25,26 The topographic and friction images were taken at an applied force ∼1 nN. For adhesive force mapping, the maximum repulsive force applied to the sample surface was ∼2 nN. Lighter levels in the images correspond to higher height (a), higher friction (b), and higher adhesion (c). The two phases had a mean surface roughness of ∼1 Å (over approximately 4 µm2 areas). A cross section taken along the line indicated by the arrows in the topographic image is shown beneath (a).

elastic model, the apparent thickness of the DOPE layer does not change significantly with the applied load, suggesting the film is inelastically deformed as a result of low intermolecular cohesive forces. The thicknesses measured for the two lipids are significantly smaller than the space-filling thicknesses. To determine the thicknesses of the lipid films under minimal load, the cut monolayers were imaged using

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Figure 4. Monolayer thicknesses measured by AFM as a function of the 2/3 power of the externally applied force F. Bars are the standard deviation of five measurements.

tapping mode. These thicknesses are found to be 3.0 ( 0.1 nm for DSPE and 2.4 ( 0.1 nm for DOPE, which is, for DSPE, in good agreement with the prediction made from the volume and area of the molecules at the airwater interface.31 The thicknesses obtained in tapping mode are ∼0.3 nm (DSPE) and ∼0.5 nm (DOPE) shorter than the space-filling thicknesses suggesting that the two phospholipids assume a tilted orientation in the supported monolayer, the tilt being more pronounced for DOPE. Comparison of the step height of the two lipid phases in contact (∼1.3 nm) and tapping (∼0.6 nm) modes indicates that the contact mode imaging forces cause a relative deformation of DOPE film vs DSPE film of ∼0.7 nm. From these data, it appears that three factors contribute to the topographic contrast obtained in contact mode: (i) the length of the phospholipids, responsible for a height difference between the two phases of ∼0.4 nm, (ii) the tilt of the two phospholipids in each phase, accounting for a ∼0.2 nm increase in step height, and finally (iii) the mechanical properties of the two phases, producing an increase of ∼0.7 nm in the apparent height difference. The force-distance curves recorded over the DOPE and DSPE phases are shown in Figure 5. Large hysteresis upon retraction can be seen, indicative of strong adhesive forces between the probe and the film. The adhesion pulloff forces over DOPE (10.5 ( 0.2 nN)33 are clearly larger than those over DSPE (6.1 ( 0.2 nN), which is also shown in the adhesion map in Figure 3c. The difference in adhesion is directly correlated to the friction contrast (Figure 3b). Surface energy does not appear to account for the adhesion contrast as both lipids are methylterminated. Rather, we attribute the difference in adhesion to the mechanical responses of the two lipid phases, i.e., the inelastic deformation of a monolayer film by a probe has been shown to increase the probe-sample contact area, resulting in a larger adhesive force.34 (31) The monolayer thickness of DSPE can be estimated by dividing the volume of the molecule by the area (41 Å2/molecule) occupied at the air-water interface at the deposition pressure. Assuming a volume for a saturated chain in the solid state of 27.4 + 26.9n Å3 per n-carbon chain32 and a head group volume of 243 Å3 gives a monolayer thickness of 3.0 nm. Note that such calculation cannot be done accurately for DOPE due to its unknown density. (32) Marra, J.; Israelachvili, J. Biochemistry 1985, 24, 4608. (33) Mean value and standard deviation of 10 pull-off forces measured in three different regions. The absolute values quoted for the adhesive forces can vary from one experiment to another due to changes in probe chemistry and geometry. (34) Weihs, T. P.; Nawaz, Z.; Jarvis, S. P.; Pethica, J. B. Appl. Phys. Lett. 1991, 59, 3536.

Figure 5. Typical force-piezo displacement curves measured on a mixed DSPE/DOPE monolayer in air: (a) DOPE phase and (b) DSPE phase. The curve for one approach/retraction cycle are made of 1024 data points, the circles representing every 16th data point. Note that the piezo displacement is not the actual probe-sample separation distance. The spring constant of the cantilever was 0.03 N/m. The softness of the cantilever required large piezo displacements in order to measure adhesion. The maximum repulsive force applied to the sample surface was ∼2 nN.

Two mechanisms may be responsible for the observed difference in friction. First, the friction behavior can be related to the relative order of the two lipid phases. In a recent study of self-assembled monolayers, Xiao and co-workers35 attributed higher friction in less ordered short chains to a larger number of energy dissipation modes. The molecular disorder of DOPE caused by cis-double bonds in the hydrocarbon tails may increase the number of dissipative modes in the molecules and give rise to higher friction. Second, friction is also known to scale with contact area.36 Therefore, the increased probe-sample contact area for the DOPE phase resulting from a deeper penetration of the probe may be responsible for higher frictional forces. Taken together, the above results show that the contrasts in topography, adhesion, and friction all depend on the mechanical behavior of the two phospholipid phases. Bilayers in Water. The topographic, friction, and adhesive images of a DSPE/DOPE monolayer deposited on DSPE-coated mica in water (drawn in Figure 1b) are shown in Figure 6. As opposed to the monolayers in air, three discrete topographic levels are observed,37 the area fraction of the high (white), medium (gray), and low (black) levels being 43 ( 2, 46 ( 4, and 11 ( 2%, respectively. From the area fractions and the relative heights, it appears that the high (white) level represents DSPE domains while the two lower levels are associated with DOPE. Although some variation was found from one sample to another, the size of the DSPE domains ranged between 2 and 10 µm. Compared to the elliptical shapes of the (35) Xiao, X.; Hu, J.; Charych, D. H.; Salmeron, M. Langmuir 1996, 12, 235. (36) Bhushan, B. Handbook of Micro/Nano Tribology; CRC Press: Boca Raton, FL, 1995. (37) At forces ∼1 nN, images of the same area could be obtained without altering the sample. At large imaging forces (>5 nN) a decreased topographic contrast could sometimes be observed. Various scan speeds were tested (0.5-10 Hz, corresponding to 15-300 µm/s) and were found to have no influence on the topographic and friction contrasts. Trace and retrace images were inverted for friction and identical for topography, indicating no significant contribution of lateral forces to the apparent topographic contrast.

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Figure 7. Typical force-piezo displacement curves measured on a mixed DSPE/DOPE monolayer on DSPE-coated mica in water: (a) DOPE phase and (b, c) DSPE phase. The circles represent every 16th data point. The spring constants of the cantilevers were 0.03 N/m (a, b) and 0.5 N/m (c) and the maximum repulsive forces applied to the sample surface were ∼2 nN (a, b) and ∼6 nN (c).

Figure 6. Gray scale images (15 µm × 15 µm) of a mixed DSPE/DOPE monolayer on DSPE-coated mica in triply-distilled water: (a) topography (z-range: 40 nm), (b) friction, and (c) adhesive force. The topographic and friction images were taken at an applied force ∼1 nN. For adhesive force mapping, the maximum repulsive force applied to the sample surface was ∼2 nN. Lighter levels in the images correspond to higher height (a), higher friction (b), and higher adhesion (c). The mean surface roughness of each phase was ∼2 Å.

monolayers, the DSPE domains in the bilayer system have a more complex, elongated shape. This change in domain shape may originate from the presence of competing interactions in the film, i.e., when line tension predominates, circular domain shapes are favored because this minimizes the length of the domain boundary. However, if repulsive interactions between individual electric dipoles become important, as could be expected with phospholipids, elongated and branched shapes are known to form.38 The shape and relative height of the lower (black) domains suggest they are composed of structural defects (38) Seul, M.; Andelman, D. Science 1995, 267, 476. Lipp, M. M.; Lee, K. Y. C.; Zasadzinski, J. A.; Waring, A. J. Science 1996, 273, 1196.

in the form of holes in the DOPE layer, in agreement with a previous study on the stability of unsaturated phospholipid bilayers.11 The depth of these holes provides an estimation of the DOPE layer thickness, 1.4 ( 0.4 nm, which is similar to that found for the monolayers in air in contact mode. Strikingly, the step height measured between the DSPE phase and the DOPE phase is 4.8 ( 0.7 nm, much larger than that obtained for the monolayers in air. Clearly, this observation cannot be accounted for by a simple difference in molecular length, tilt, and film deformation. In the following sections, we provide evidence that this high contrast in the topographic image originates from a difference in surface forces over the two lipid phases. Similar surface forces would be expected over the DSPE and DOPE phases since they have identical hydrophilic head groups. However, the force-distance curves show that this is clearly not the case. First, during the approach a snap to contact indicative of attractive van der Waals interactions is found for the DOPE surface (Figure 7a), but is lacking for DSPE (Figure 7b). Second, upon retraction the DOPE phase shows a strong adhesive pulloff force (4.7 ( 0.2 nN), while there is almost no adhesion on the DSPE phase, yielding a highly contrasted adhesion image (Figure 6c). These two features in the force curves indicate that the van der Waals interactions between the DSPE surface and the probe are offset by a strong repulsive force. Further evidence of a short-range repulsion is observed when the maximum applied force is increased to greater than 4 nN. While the forces over the DOPE regions remain

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mechanical stability under the studied loads. While the DSPE film has strong cohesive forces due to strong lateral interactions between the molecules, a lower cohesion/ stability is expected for DOPE due to reduced van der Waals interactions between the unsaturated hydrocarbon chains and reduced possibility for hydrogen bonds between the head groups. Therefore, at short separation distances the minimum force exerted by the AFM probe on the DOPE film may cause a disruption of the lipid head groups and of the hydration layer. This finding, that hydration/steric repulsion over lipid bilayers depends on their mechanical stability, is of biological relevance in the control of cell adhesion and membrane fusion processes. The image contrast mechanism can now be understood in the light of these findings, as illustrated in Figure 8. It is clear that the topography, adhesion, and friction of the two phospholipid phases are imaged in two different regimes.41 The low imaging force used in this study is large enough for the probe to establish contact with the DOPE surface and indent ∼1 nm into the film, whereas at the same load the hydration/steric repulsion over the DSPE phase cannot be overcome, thus keeping the probe ∼3 nm off the head group surface. Accordingly, the differences in mechanical properties and surface forces of the two phospholipid phases account for the increased topographic contrast. Figure 8. Schematic drawing showing the two different imaging regimes over the DSPE/DOPE bilayer in water. While the DSPE phase is imaged with the probe ∼3 nm off the head group surface (a), the DOPE phase is compressed over a depth of ∼1 nm during imaging (b).

unchanged, a completely different force is obtained over the DSPE regions (Figure 7c). At a surface separation of ∼3 nm, a steep repulsion can be seen, where the force increases until the probe jumps to contact, indicating a change from a net repulsive to net attractive force and that the gradient of the force between the probe and surface has exceeded the spring constant of the cantilever. This repulsion decays exponentially with distance with a decay length of ∼0.7 nm and is indicative of hydration/steric interaction forces, which is consistent with data obtained with the osmotic stress (OS) technique39 and the surface forces apparatus (SFA)32 on lipid bilayers. A snap to contact at a critical load was not observed in the OS and SFA studies; however, the pressure applied in the present study at 4 nN is 7 orders of magnitude greater than the pressure used in OS experiments and is the upper limit of the pressure that can be accessed with the SFA.40 The snap to contact suggests that the DSPE head groups are deformed by ∼4 nN loads, resulting in the disruption of the structure of the water layer. The unexpected apparent lack of hydration/steric forces over the DOPE film appears to be related to its low (39) Rand, R. P.; Parsegian, V. A. Biochim. Biophys. Acta 1989, 988, 351. (40) An accurate estimate of the load exerted on the hydrated DSPE surface at the snap to contact would require a knowledge of the “mechanical properties” of the hydration layer. Lacking this information we can estimate a lower limit of pressure of ∼3 MPa, by assuming that the 4 nN force is distributed over the entire probe area (∼20 nm radius). The actual pressure exerted by the apex of the probe is much larger than this lower limit.

Conclusions Mixed phospholipid monolayers and bilayers exhibiting phase separation have been used to define the probesample separation distance while imaging their surface properties by AFM. Three factors contribute to the topographic contrast of the monolayers in air: (i) the length of the phospholipids, (ii) the tilt assumed by the phospholipids in each phase, and most importantly (iii) the relative mechanical properties of the lipid phases, the latter factor being also responsible for adhesion and friction contrasts. For bilayers in water, a fourth parameter is found to determine the contrast in topography, adhesion, and friction, i.e. short-range repulsive hydration/steric forces. This study demonstrates the important role that mechanical properties play in the surface behavior of phospholipid films and emphasizes the potential of AFM for investigating the properties of cell membranes in medicine and biology. For instance, cell adhesion molecules such as glycolipids can now be incorporated into a heterogeneous lipid bilayer membrane and studied by AFM. Acknowledgment. This research was supported by the Office of Naval Research (ONR) and by a NATO Research Fellowship (Y. F. Dufreˆne). We also thank M. Fletcher, R. J. Colton, N. A. Burnham, J. Jones-Meehan, and the members of NRL Code 6177 for valuable discussions, K. Lee for programming associated with the adhesion images, and J. Callahan and M. Shahgholi for electrospray mass spectrometry. LA970221R (41) Note that two modes of imaging based on repulsive interactions have been recently reported. Senden, T. J.; Drummond, C. J.; Ke´kicheff, P. Langmuir 1994, 10, 358. Manne, S.; Cleveland, J. P.; Gaub, H. E.; Stucky, G. D.; Hansma, P. K. Langmuir 1994, 10, 4409.