Langmuir 2000, 16, 1481-1484
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Imaging of Domain Structures in a One-Component Lipid Monolayer by Time-of-Flight Secondary Ion Mass Spectrometry Nikolaus Bourdos,† Felix Kollmer,‡ Alfred Benninghoven,‡ Manfred Sieber,† and Hans-Joachim Galla*,† Institut fu¨ r Biochemie, Westfa¨ lische Wilhelms-Universita¨ t Mu¨ nster, Wilhelm-Klemm-Strasse 2, D-48149 Mu¨ nster, Germany, and Physikalisches Institut, Westfa¨ lische Wilhelms-Universita¨ t Mu¨ nster, Wilhelm-Klemm-Strasse 10, D-48149 Mu¨ nster, Germany Received September 27, 1999. In Final Form: December 20, 1999 Time-of-flight secondary ion mass spectrometry with high lateral resolution was used to image the domain structures of a phase-separated pure dipalmitoylphosphatidylcholine monolayer, which was transferred from the air-water interface of a Langmuir film balance to a gold surface. In the two-phase region characteristic domain structures became clearly visible in the image maps of different fragment ions (secondary ions) as well as the molecule ion. Depending on the fragment, the liquid-condensed (LC) domains exhibit both lower and higher secondary ion yields than the liquid-expanded regions of the monolayer. To understand the mechanism leading to this alternating contrast, we determined the intensities of some secondary ions at different surface pressures adjusted during transfer. This analysis showed that the secondary ion formation in the lipid headgroup region is much more sensitive to the physical state of the monolayer than in the acyl chains. We conclude that an increased electrostatic interaction among the closer packed molecules in the LC phase accounts for the lower yield of headgroup fragments. The observed increased yields in the LC phase leading to a negative contrast may be due to a more fundamental mechanism of formation of the respective secondary ion like weakening of the chemical bonds or favoring proton transfer.
It is well-known that monolayers of lipids and fatty acids at the air-water interface reveal a complex phase behavior.1,2 In the region of coexistence of the liquidexpanded (LE) and liquid-condensed (LC) phase, characteristic domain structures can be visualized by fluorescence light microscopy (FLM) or brewster angle microscopy (BAM).3,4 Although being valuable tools for structural analysis, these techniques do not yield information about the composition of the different phases. A quantitative analysis of the micrographs is therefore limited to one-component systems. Conventional mass spectrometry provides substantial information about the chemical structure but could not be applied to determine the lateral distribution of a molecular species in a monolayer. This disadvantage of the otherwise powerful method was compensated by the introduction of time-of-flight secondary ion mass spectrometry (TOF-SIMS) where ionization and desorption of the target molecules are achieved by an ion beam which is precisely positioned and focused on the sample surface. Equipped with a scanning unit, TOF-SIMS is suited to produce laterally resolved images of the secondary ion distribution. Since the ionization process is limited to the uppermost layers of the sample, TOF-SIMS is well-suited to investigate solid-supported monolayers of amphiphilic substances. In a former study5 we showed that in a twocomponent lipid monolayer domain structures can be * To whom correspondence should be addressed. † Institut fu ¨ r Biochemie. ‡ Physikalisches Institut. (1) Albrecht, O.; Gruler, H.; Sackmann, E. J. Phys. (Paris) 1978, 39, 301. (2) McConnell, H. M.; Tamm, L. K.; Weis, R. M. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 3249. (3) Lo¨sche, M.; Sackmann, E.; Mo¨hwald, H. Ber. Bunsen-Ges. Phys. Chem. 1983, 87, 848. (4) McConlogue, C. W.; Vanderlick, T. K. Langmuir 1997, 13, 7158. (5) Leufgen, K. M.; Rulle, H.; Benninghoven, A.; Sieber, M. Langmuir 1996, 12, 1708.
visualized by TOF-SIMS due to their different chemical composition. In the present study we report the visualization of domain structures in the biphasic state of a onecomponent system. Contrast in the images is thus caused only by the differing physical states of the lipids in the two phases. Information about the mechanisms underlying the observed contrast is gained from the dependence of the secondary ion intensities on the surface density of the molecules. Solid-supported monolayers of L-R-dipalmitoylphosphatidylcholine (DPPC) were prepared on gold-covered glass slides, serving as substrates onto which the DPPC films are transferred, by first evaporating a 1 nm layer of chromium followed by a gold layer of 200 nm thickness at a rate of 1 nm/s. The chromium serves as an adhesive layer for the gold which does not stick on pure glass surfaces directly. After evaporation, the slides were rectified in a Soxhlet for 8 h with n-hexane and exposed to argon plasma for 3 min to yield hydrophobic gold surfaces.6 Hydrophobic gold as substrate material was necessary to guarantee a nonreactive transfer which preserves domain structures in films with coexisting phases (unpublished data). The topography of the gold surfaces was checked with scanning force microscopy exhibiting a roughness with grains of about 20 nm diameter and height around 1.7 nm. The DPPC was spread from a chloroform solution at 20 °C onto the air-water interface of a Langmuir film balance. After solvent evaporation films were compressed to the corresponding surface pressures (or molecular areas AM), indicated by the arrows in Figure 1, and equilibrating them in a constant-pressure mode. The monolayers were transferred to the slides using the Langmuir-Blodgett (LB) technique. Preparations in the two-phase region were carried out by (6) Ulman, A. An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self-Assembly; Academic Press: San Diego, CA, 1991; p 109.
10.1021/la9912693 CCC: $19.00 © 2000 American Chemical Society Published on Web 01/29/2000
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Figure 1. Dependence of the surface pressure on the area per molecule of a DPPC monolayer spread at the air/water interface of a film balance at 20 °C. To analyze the monolayer by TOFSIMS, the film was transferred to solid substrates at areas per molecule indicated by arrows.
Figure 2. Mass spectrum of a DPPC monolayer transferred to gold at π ) 5 mN/m. The most important fragmentations are indicated in the inset.
compressing the DPPC monolayer at a fixed speed (0.05 nm2‚molecule-1‚min-1) and halting the barrier at the desired point of the isotherm. Due to the high compressibility of the monolayer in the two-phase region, the transfer could only be carried out in the constant-area mode. Thus the lipid-covered surface of the trough was reduced simultaneously during transfer by an amount equal to the area of the transferred monolayer. The transferred monolayers were analyzed with TOFSIMS by scanning a sample area of 120 × 120 µm2 (imaging, see Figure 3) and 60 × 60 µm2 (intensity analysis, see Figure 4). For imaging, a 512 × 512 pixel raster was chosen for best lateral resolution. Primary ions were provided by a gallium liquid metal ion source. Due to the size of the imaged area, the lateral resolution in this study is limited by the pixel raster to ≈230 nm, whereas the physical limit is determined by the minimal diameter of the Ga+ beam which is about ≈80 nm. This has to be taken into account if smaller areas are scanned at the same pixel resolution. The maximal lateral resolution that could be attained theoretically was ≈10 nm, calculated for the secondary ion C3H8N+ (mass M ) 58) from its secondary ion yield and damage cross section.7 The mass spectrum of positive secondary ions, obtained after integration over the scanned area together with a fragmentation scheme, is shown in Figure 2. In the region of high molecular masses the protonated (MDPPC+H ) 735) and dehydrogenated (MDPPC-H ) 733) DPPC ion (“quasi(7) Ko¨tter, F.; Benninghoven, A. Appl. Surf. Sci. 1998, 133, 47.
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molecule ion”) could be identified. In the region of intermediate masses, signals of the substrate surface (MAu ) 197) and intense headgroup fragments such as phosphocholine (PC; M ) 184), choline (M ) 104), and choline minus OH (M ) 86) are clearly visible. The region of small masses is mainly governed by hydrocarbon fragments, whose origin can be assigned to the acyl chains. This fragmentation largely corresponds to the one obtained by other mass spectrometric methods.8 The applied primary ion dose density (number of ions per unit area), a measure for the extent of reduction of the layer by sputtering, was 1013 ions/cm2. Figure 3 shows images of a gold-supported DPPC monolayer in the two-phase region, which are obtained by mapping the detected intensities of the signals of a given secondary ion. Images of most of the fragment ions as well as the molecule ion itself exhibit considerable contrast. Clearly, domain structures with the shape and size as found by microscopic techniques are visible: kidney-shaped domains of the LC phase are embedded in the LE phase. The relative area occupied by the LC phase is in good agreement with the extent of the phase separation defined by the value of the molecular area during LB transfer. Generally, the secondary ions generated in the LE-phase monolayer showed higher signal intensities which will be called positive contrast in the following. Some of the fragment ions, such as those with a mass-to-charge ratio of 27, 29, or 104, and the gold ion (MAu ) 197) yielded higher signal intensity in the LCphase region of the monolayer which we call negative contrast. Due to the higher surface density of the lipids in the LC phase, positive contrast can only be explained by an influence of the physical state of the lipid on the secondary ion yield. We further elucidated the influence of the physical state of the lipid monolayer domain on the secondary ion yield. Monolayers of DPPC were transferred at a given molecular area from the air-water interface to a gold substrate, and mass spectra were taken from each sample and analyzed with respect to intensity. One has to consider, however, that absolute peak intensities are sensitive to the surface properties of the solid support (roughness, purity) and second that by changing the molecular surface density the number of molecules in the focus of the primary ion beam will change. Therefore we had to correct the raw signal intensity by the intensity of a secondary ion which is independent of the physical state of the monolayer. To determine the best suited reference secondary ion, the raw intensities Iraw were divided by the surface concentration Γ ) 1/AM and plotted versus the molecular area (data not shown). This analysis showed that the ionization and desorption of the small hydrocarbons from the acyl chains were nearly independent of the surface density and thus on the physical state of the monolayer. Especially the secondary ions CH3+, C2H3+, and C2H5+ were well suited since they only showed variations due to differences in the physical properties of the solid supports but not due to the monolayer properties. From these possible fragment ions C2H3+ (M ) 27) was chosen as a reference because of its high signal intensity. The corrected signal intensities
IM ) Iraw/Iraw,27 are then proportional to the intensities per molecule and (8) Ayanoglu, E.; Wegmann, A.; Pilet, O.; Marbury, G. D.; Hass, J. R.; Djerassi, C. J. Am. Chem. Soc. 1984, 106, 5246.
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Figure 3. Image maps evaluated from secondary ion mass spectra of phase-separated monolayers (constant area mode at 0.7 nm2). Each image map represents the lateral distribution of a particular fragment ion (a, M ) 58; b, M ) 104; c, M ) 166; d, M ) 735).
Figure 4. Dependence of the relative, corrected signal intensities I* ) IM/IM,AM ) 0.96 nm2 of some characteristic fragment ions on the area per molecule adjusted during transfer. The two-phase region of the system is indicated by vertical lines.
therefore independent of the surface density and the properties of the solid support. Figure 4 depicts the dependence of the corrected signal intensities IM of some characteristic secondary ions and the molecule ion on the molecular area. To simplify the graphical presentation, the corrected intensities were normalized to one at the intensity of AM ) 0.96 nm2. From Figure 4 it follows that in general all the secondary ion intensities decrease as the molecular area decreases (or surface density increases). In the LE phase this conclusion is not strictly verified since intensities were determined only at two different molecular areas, but the tendency was the same. In the two-phase region intensities showed a linear decrease with increasing amount of LC phase lipids or remained constant (M ) 43). In some cases the intensities of the fragment ions of the LC phase lipids were higher, leading to the negative contrast observed in the corresponding images (Figure 3, M ) 104). The nearly linear course in the LE/LC coexistence range indicates that the corresponding secondary ion intensities of the lipids of both phases simply sum up. In the LC phase, intensities declined more exponentially to a limit with decreasing molecular area. This limit seems to represent the intensity of secondary ions originating from a tightly packed, crystalline monolayer of lipids. With respect to the corrected signal intensity IM in the pure LE phase at a molecular area of 0.96 nm2, the small hydrocarbons exhibited the smallest total decline of their intensities by about 30%, whereas the intensity of the quasi-molecule ion (IM)735) declined by 99%. Intensities of some secondary ions of the lipid headgroup also showed a strong dependence from the physical state, like the PC minus H2O fragment (M ) 166), which declined by nearly 80%.
From different studies on lipid mono- and multilayers, it is known that the acyl chains of the lipids exhibit a different degree of lateral order in the respective phases. Order may change along one acyl chain or between acyl chains of neighboring lipids. It was demonstrated by several techniques such as NMR spectroscopy and by molecular dynamics simulations9-11 that the methyl and ethyl groups at the end of the acyl chains are less sensitive to changes of external variables such as lateral pressure or temperature. This may explain the lower decline of the intensities of the corresponding secondary hydrocarbon ions. Dicko et al. recently showed by IR spectroscopy using dipalmitoylphosphatidylglycerol (DPPG) monolayers on a solid support and at the air/water interface that not only the acyl chains undergo changes in conformation and orientation but the conformation of the headgroups changes as well upon compression.12 Moreover, in a transferred film the conformation of the headgroup changes due to dehydration, and the acyl chains were found to be more ordered. Of course, dehydration as a consequence (or artifact) of transfer may be due to hydrophilicity of the substrate13 since there is a strong affinity between the polar headgroup and the substrate, if the head-down transfer is reactive. This would explain the findings of Dicko et al., who prepared the DPPG monolayers on germanium. Whether a change of conformation influences the respective secondary ion yields of the headgroup fragments that strongly, cannot be stated from our study. Considering the hydrocarbon ions, the yields are lower in the higherordered LC phase, but in the case of the choline secondary ion (M ) 104), yields are increased in the LC phase. The tighter packing of the molecules in the LC state may cause two effects. A more pronounced electrostatic interaction among the headgroups may be present, causing smaller yields due to the higher energy needed to hurl out a molecule from the layer. On the other hand, a smaller distance can alter polarization of chemical bonds and/or favor proton transfer. Note that the choline ion (see the insert in Figure 2) cannot be formed by simple cleavage from the PC moiety leading to a zwitterionic choline minus H. Rather a proton has to be transferred to produce a positive choline fragment. The opposite contrasts for, e.g., the secondary ions M ) 104 and M ) 166, clearly demonstrate that secondary ion distributions are not a direct measure of the physical state of the system under investigation. Actual local surface concentration of a (9) Seelig, A.; Seelig, J. Biochemistry 1974, 13, 4839. (10) Venable, R. M.; Zhang, Y.; Hardy, B. J.; Pastor, R. W. Science 1993, 262, 223. (11) Petrache, H. I.; Tu, K.; Nagle, J. F. Biophys. J. 1999, 76, 2479. (12) Dicko, A.; Bourque, H.; Pe`zolet, M. Chem. Phys. Lipids 1998, 96, 125. (13) Petrov, J. G.; Kuhn, H.; Mo¨bius, D. J. Colloid Interface Sci. 1980, 73, 66.
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molecule in a monolayer is not derived directly from the respective secondary ion yield. This has been shown here in a one-component system and has to be considered especially in more complicated systems with a larger number of constituents or more complex molecules such as proteins. In conclusion, the present study shows that TOF-SIMS is well suited to study changes in the physical state of molecules in monomolecular overlayers such as LB films. Contrast in TOF-SIMS images is generated not only by chemical but also by physical differences within the film
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of the substrate. By a quantitative evaluation of the spectra and images these differences may be separated from each other, which allows study of more complex systems like protein-containing reconstituted lipid monolayers or even biological membranes. Acknowledgment. This work was supported by the Deutsche Forschungsgemeinschaft, GA 233/18-1. LA9912693
