Langmuir 1992,8, 1768-1774
1768
Molecular Order in Polymerizable Langmuir-Blodget t Films Probed by Microfluorescence and Scanning Force Microscopy Barbara M. Goettgens,+Ralf W. Tillmann, Manfred Radmacher, and Hermann E. Gaub' Physics Department, Technical University Munich, 8046 Garching, Germany Received January 7, 1992. In Final Form: March 9,1992 Films of a polymerizable fatty acid (lO,l2-pentacosadiynoicacid) containing diacetylenic groups were investigated by microfluorescence, film balance techniques, and scanning force microscopy. At the airwater interface the fatty acid forms films whose properties can be controlled via the pH of the subphase. We found that under certain conditions a solid-solid transition occurs which results in two phases with different fluorescence properties of the polymer. The structure of these films was investigated by scanning force microscopy. Images at molecular resolution of both monomeric and polymeric films are reported here for the first time. The fatty acids exhibited in both cases long-range positional order with comparable lattice constants. Introduction Lipid films on solid supports are gaining increasing attention not only as model systems for cell membrane~,l-~ but also for their technological applications as novel m a t e ~ i a l s .Often, ~ ~ ~ however, their inherent flexibility and lateral mobility are disadvantageous. For certain applications a higher rigidity would be desirable. One attempt to increase the mechanical stability of such films is to chemically cross-linkthe lipids. This has become possible by the introduction of various polymerizable groups in the lipids. During the last decade the synthesis of a variety of such polymerizable lipids by several groups has lead to a rich selection of suitable molecules.6J Among the most popular polymerizable groups are the diacetylenes. After cross-linking, their polymer backbone exhibits unique optical properties, which to a certain degree, allow the characterization of the polymer by spectroscopical techniques.819 Because of their durability, polymerized lipid films have also been among the first organic systems to be imaged with the scanning force microscope (SFM).'@l4 In this study here, we investigate the molecular structure of ~~
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* To whom correspondence should be addressed.
+ Present address: Siemens-NixdorfAG, Otto-Hahn-Ring,8000 Miinchen, Germany.
(1) Watts, T. H.; Gaub, H. E.; McConnell, H. M. Nature 1986, 320, 179-181. (2) Thompson, N. L.; Palmer, A. G. Comments Mol. Cell Biophys. 1988,5, 39-56. (3) McConnell, H. M.; Watts, T. H.; Weis, R. M.; Brian, A. A. Biochim. Biophys. Acta 1986,864,95-106. (4) Swalen, J. D.; Allara, D. L.; Andrade, J. D.; Chandross, E. A.; Ga-
roff, s.;Israelachvili, J.; McCarthy, J. G.; Murray,R.; Peaae,R. F.;Rabold, J. F.; Wynne, K. J.; Yu, H. Langmuir 1987, 3, 932. ( 6 ) Schuhmann, W.; Heyn, S. P.; Gaub, H. E. Adu. Mater. 1991, 3, 388-391.
(6) Bader, H.; Dorn, K.; Hupfer, B.; Ringsdorf, H. In Polymer Membranes; M. Gordon, Ed.; Springer: Berlin, Heidelberg, New York, Tokyo, 1985; pp 2-62. (7) Gaub, H. E.; Sackmann, E.; Biischl, R.; Ringsdorf, H. Biophys. J. 1984,45, 725-731.
films from a polymerizable diacetylenic fatty acid by film balance techniques, by microfluorescence, and by scanning force microscopy in both its monomeric and its polymeric form.
Materials and Methods Lipids. 10,12-Pentacosadiynoicacid (PCA)(seeFigure 1)was purchased from Farchan Laboratories,Karlsruhe,FRG, and recrystallized several times in 5:l hexane/ethanol (Merck, Darmstadt, FRG). The purity was confirmed by thin-layer chromatography. For the fluorescence film balance studies of nonpolymerized films, the fluorophorN-(TexasRed sulfony1)dipalmitoyl-L-a-phosphatidylethanolamine(TxRed-PE) (Molecular Probes, Eugene, OR) was added to the PCA at a concentration of 0.1 mol %. Fluorescence Film Balance. A modular microfluorescence film balance built in our laboratory was used. The pressurearea diagrams were recorded with a largetrough (340cm2)which was also used for Langmuir-Blodgett (LB) deposition, whereas the microfluorescence studies were performed with a small through (31cm2). The experimental setup is described in detail by Heyn et al.15 As subphase,either pure water (Milli-Qsystem) or one of the following buffers was used: at pH 4.7,lO mM citric acid plus 10mM NaCl; at pH 7.6 8.0,lO mM Hepes plus 10 mM NaCl;at pH 9.5,lO mM glycine plus 10mM NaCl; for LB coating, 0.5 mM CdC12 in 10 mM Hepes at pH 7.5. The corresponding pH values were titrated by NaOH. Polymerization of the films was carried out at the air-water interface by UV irradiation (Hg pen ray, LOT, Darmstadt, FRG) for 30 8 . During polymerization the area was kept constant. Scanning Force Microscopy. SFM was performed with a home-built instrumentdescribed in detailelsewhere.16 The device is a combination of an epifluorescence microscope and a SFM (see Figure 2). This combination allows one to position the tip guided by the lightmicroscope,e.g., on top of a fluorescentdomain, prior to zooming in by SFM down to molecular resolution. It also offers the possibilityto locally characterizethe fluorescence in terms of polarization or spectroscopical properties. The instrument allows in-parallel imaging by microfluorescence in the range 1000-2 pm and by SFM in the range from 15pm down to atomic dimensions. It thus covers more than 7 orders of
(8) Warta, R.; Sixl, H. J. Chem. Phys. 1988,88, 95-99. (9) Bubek, C.; Tieke, B.; Wegner, G. Ber. Bunsen-Ges. Phys. Chem. 1982,86,495-498. (10) Marti,O.;Ribi,H.;Drake,B.;Albrecht,T.R.;Quate,C.F.;Hansma, (13) Hansma, H. E.; Weisenhorn, A. L.; Edmundson, A. B.; Gaub, H. P. K. Science 1988,239,50-52. E.; Hansma, P. K. Clin. Chem. 1991,37 (9), 1497-1501. (11) Radmacher, M.; Goettgens, B. M.; Tillmann, R. W.; Hansama, H. (14) Yamada, H.; Akamine, S.; Quate, C. F.Ultramicroscopy, in press
G.; Hansma, P. K.; Gaub, H. E. In Scanned Probe Microscopies; Wickramasinghe, K., McDonald, F. A,, Eds.; American Institute of Physics: New York 1991. (12) Weisenhorn, A.; Gaub, H. E.; Hansma, H. G.; Sinsheimer, R. L.; Keldermann, G. L.; Hansma, P. Scanning Microsc. 1990, 3, 511-516.
(Mai).
(15) Heyn, S. P.; Tillmann, R. W.; Egger, M.; Gaub, H. E. J.Biochem. Biophys. Methods 1990,22, 145-158. (16) Radmacher, M.; Eberle, K.; Gaub, H. E. Ultramicroscopy, in press (Mai).
0743-746319212408-1768$03.0010 0 1992 American Chemical Society
Langmuir, Vol. 8, No. 7, 1992 1769
Molecular Order in Polymerizable LB Films
Figure 1. Schematics of a film from 10,12-pentacosadiynoic acid (PCA) and its topochemical polymerization by UV irradiation.
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Figure 3. (a) Fluorescence micrograph of monomeric PCA on Millipore at a pressure of 2 mN/m. (b) Multilayer formation of the same film at 10 mN/m.
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Figure 2. Schematics of the microfluorescence/scanning force
microscope. The SFM setup consists of a conventional tube scanner driven by a microcomputer and an optical detection sensor for the cantilever deflection. The lens which focuses the laser onto the backside of the cantilever for the deflection detection is in this setup a microscope objective lens which is integrated into the SFM. This lens serves at the same time as the objective for the microfluorescence setup with epifluorescence excitation. As Abbe optics is used, both instruments are mechanically decoupled which is of great importance for the resolution of the SFM.
magnitude in magnification. For SFM imaging, commercially availablesilicon nitride cantilevers (Park Scientific Instruments, Mountain View, CA)with integrated tips were used and routinely cleaned by Ar-plasma discharge.
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Figure 4. Pressurearea isotherms (7' = 20 "C) of PCA as a function of the pH. Substrates. (100)p-typesilicon waferswitha thermdygrown
oxide layer of about 150-nm thickness (a kind gift of Wacker
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Goettgens et aZ.
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Possible Orientations
of the Polymer Backbone
Figure 5. (a) and (b) Fluorescence micrograph of PCA domains polymerized at p = 4 mN/m on Hepes (pH 9) and T = 20 "C for differentexcitation polarizations(excitationwavelength 580 nm) (1bar = 30 pm). (c) Molecular model for the low-pressure configurationsof the diacetylenic groups and the two possible polymer orientations. Burghausen, FRG) were cleaned by ultrasonicationin 2 % Hellmanex (Hellma, Miillheim, FRG). The wafers were then rinsed extensively with water (Milli-Q system, Molsheim, France) and finally with methanol (HPLC quality,Aldrich, Milwaukee, WI). When images were taken in air, the fatty acid films were transferred by conventionalverticalpulling at constant pressure. For investigations under water, the wafers were precoated at p = 25 mN/m with a monolayer of the polymerized Cd salt of PCA as described above. Then the monolayer to be investigated was transferred by horizontal dipping17and kept submerged.
Results and Discussion
As is well known, amphiphilic molecules at the airwater interface offer a richness of two-dimensional phases. The molecules used here have the unique property of being polymerizable (See Figure l),provided their diacetylenic groups are in a proper orientation and relative distance with respect to each other. This requirement is fulfilled only in solid phases. For our investigations with the microfluorescence film balance as well as later on with the SFM, it is desirable that the solid domains are well ordered and as large as possible. We therefore first investigated several strategies for the controlled growth of single crystals from PCA at the air-water interface. The lowest curve in Figure 4 shows the well-known pressure-area isotherm of PCA at room temperature on water. It exhibits a short steep pressure increase at about 23 A2 per molecule which is followed by a more or less flat regime and a second steep increase at about 8A2per molecule. The first pressure increaseoccurs at a molecular area which corresponds nicely to a tight packing of the molecules whereas the second pressure increase has to be attributed to a multilayer formation. This can be directly visualized by microfluorescence. The dark needles in Figure 3a represent solid domains, from which the traces of fluorescent lipid have been excluded. The image in Figure 3b was taken in the plateau range and shows how the monolayer was folded down into the (17) Tscharner, V.; McConnell, H. M. Biophys. J. 1981,36,421-427.
subphase. With water as subphase, the crystallization occurs at very low pressures and crystal growth is hardly controllable under these conditions. One possibility to overcome this difficulty would be to work at elevated temperatures. This would shift the onset of crystallization to higher pressures and result in a more pronounced fluid-solid coexistence regime. But due to the experimental difficulties at elevated temperatures, we decided to alter the charge density of the film instead. With increasing Coulomb repulsion between the head groups, the crystallizationrequires higher pressures,18and as a consequence, the fluid-solid coexistence should be shifted to moderate pressures. As the fatty acid has a titrable carbonyl group this can be easily done by varying the pH of the subphase. In Figure 4, the influence of the pH on the pressure-area isotherm is shown. A t a pH value of about 10, the critical point is reached, and above this value no solid phase is stable at room temperature. For further studies, we chose a pH value of 8. Here the fluidsolid coexistence occurs at pressures around 5 mN/m. Single crystals as large as fractions of millimeters could be grown under these conditions. The polymerization of the diacetylenes is a topochemical reaction. As it was pointed out already, this means that the reaction occurs only in a certain relative arrangement of the diacetylene groups which generally is guaranteed only in a crystalline arrangement (see Figure 1). Earlier studies have shown that the intrinsic fluorescence of the resulting polydiacetylenic backbone can reveal detailed information about the molecular arrangement in the solid domains.lg We have utilized this approach here to investigate the structure of our polymerized PCA domains. (18) Cevc, G.; Marsh, D. Phospholipid bilayers; Wiley: New York, 1987. (19) Gijbel, H. D.; Gaub, H. E.; Mijhwald, H. Chem. Phys. Lett. 1987, 138,441-446.
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Polymer Backbone
Figure 6. (a) Fluorescence micrograph of a PCA domain polymerized at p = 8 mN/m on Hepes (pH 9) and T = 20 "C. (b) and (c) are same as (a) but with polarized excitation (directionindicated by the bars). (d) Molecular model for the high-pressureconfiguration of the diacetylenic groups and the resulting polymer backbone.
Figure 5 shows the fluorescence micrograph of such domains which were polymerized in the middle of the coexistence plateau at a pressure of 4 mN/m. Here the bright domains represent the polymerized PCA crystals. When excited via a linear polarizer, the individual domains appear homogeneous, but the relative brightness of different domains depends on the polarization. This means that the absorption dipoles of the polymer backbones are ordered and have a fixed relation to the macroscopic shape of the domain and thus to the crystal lattice. Interestingly the excitation does not vanish completely for any orientation of the polarizer. The polarization degree P = (1" - Imin)/(Imm + Imin) was and Imin are the measured to be P = 0.2 + -0.1. Here Imm maximum and minimum emission intensities which were corrected for the b.ackground and a weak dichroism of the microscope. The polarization degree was determined as an average from several spots on several domains. However,when the domains are polymerized at a slightly higher pressure at the very end of the coexistence (p = 8 mN/m), their fluorescence excitation may be completely suppressed by rotation of the excitation polarizer. This
is shown in Figure 6. The starlike domain obviously consists of two single crystals fused at the center. Here the polarization degree is above 0.95. This shows that the polymer backbone is, in this case, highly ordered, as most probably was the crystal before it was polymerized. It also shows that the absorption dipole moment of the polymer backbone has to lie in the plane. A detailed series of experiments, not shown here, indicates that welatively narrow pressure range separates the two regimes. Crystals polymerized in the plateau of the fluid-solid coexistenceall exhibit the same polarization degree of about 0.2. The fluorescence from crystals polymerized at the end of the fluid-solid coexistence is completely polarized. It is important to note here that no significant macroscopic shape change is detectable by microfluorescenceduring compression of the monomericfilm in that pressure range. The fractal shape of the domain in Figure 6 is somewhat atypical in that it was grown too fast and therefore exhibits the fractal shape. It was chosen mainly for its beauty. From these data the question arises whether or not the lower degree of polarization in the crystal which was po-
1772 Langmuir, Vol. 8,No. 7, 1992
lymerized at lower pressures is due to a locally less well ordered packing or whether it is caused by a different crystal structure. Electron diffraction studies on multilayers from the same molecule had revealed that under certain conditions a solid-solid transition may occur.2oIt was shown that the two corresponding phases differ in the relative orientation of the polymerizable groups to each other. In the phase with the higher density these researchers found the diacetylene groups to be arranged in a herring bone pattern whereas in the phase with the lower density the packing was simply triclinic. Interestingly this differencecauses a symmetry break with respect to the potential directions in which the linear polydiacetylene can grow. The modes for these two phases are sketched in Figures 5c and 6d. In the high-density phase with the herring bone pattern only one orientation of the polymer backbone is possible, resulting in a high polarization degree. In a single crystal of the low density phase, however, two directions of the polymer backbone are equivalent. If the polymers are shorter than the resolution of our fluorescence microscope, this results in a laterally homogeneous fluorescence, but due to the two equally possible orientations,the mean polarization lays in between the two polymer directions with a polarization degree of only P = 0.268. The calculation of this value is based on the assumption that the absorption dipole moment is parallel to the polymer backbone and that the two polymer orientations include an angle of 60'. Two findings make us believe that this model describes the two phases that we have found. First, within the experimental error, the measured polarization degree is in good agreement with the calculated value, and second, we do not find a broad range of polarization degrees in the low-pressurephase upon varying the pressure. One would have to expectthe latter effect if just packing irregularities or a breakup of the crystals during polymerization in the low-pressure phase would be the cause of the low polarization degree. For further investigationsof the molecular arrangement of the PCA films, we have employed a combination of SFM and microfluorescence. SFM has already proven to be an extremely useful tool for the investigation of lipid films at the molecular 1evel.11J2J4$21-23 Figure 7 shows a PCA film on silicon oxide imaged in air as seen with this instrument by microfluorescence (a) and with a higher magnificationby SFM (b). The film had been compressed to a final value of 25 mN/m (on pure water as subphase) before UV polymerization. As has been indicated already above, the film is, under these conditions, most likely a multilayer. This conjecture is verified by the surface structure as seen by the SFM. It clearly exhibits wellpronounced extended plateaus with discrete steps of multiples of the monolayer thickness. The plateaus consist of mono- and penta- but predominantly trilayers. The plateaus are flat to within some angstroms. The steps are sharp, and their slope is determined by t h tip curvature. The structures are stable during hours of scanning except for the edges which tend to become fuzzy after extensive scanning. At high magnifications, no molecular resolution could be obtained in air on any level of the various plateaus. (20) Lieser, G.; Tieke, B.; Wegner, G. Thin Solid Films 1980,68,7790. (21) Egger, M.; Ohnesorge, F.; Weisenhorn, A.; Heyn, S. P.; Drake, B.; Prater, C. B.; Gould, S. A. C.; Hansma, P.; Gaub, H. E. J. Struct. Biol. 1990,103,89-94. (22) Weisenhorn, A.; Egger, M.; Ohnesorge, F.; Gould, S. A. C.; Heyn, S. P.; Hansma, H. G.; Sinsheimer, R. L.; Gaub, H. E.; Hansma, P. Langmuir 1991, 7, 8-12. (23) Hansma, H. G.; Gould, S. A. C.; Hansma, P. K.; Gaub, H. E.; Longo, M.; Zazadinski, J. A. N. Langmuir 1991, 7, 1051-1054.
Goettgens et al.
Figure 7. (a) Fluorescencemicrograph of a collapsed PCA film polymerized and transferred onto silicon oxide, as seen by the optical part of the microfluorescence/SFM. The size of the cantilever (dark triangle) is 200 pm. (b) Area under the tip of the cantilever as probed by the SFM.
We attribute this to the high scanning forces which are inevitable when imaging in air.24 In order to investigate the molecular order in the PCA films, all further experiments were carried out on monomolecular films on hydrophobic supports. In order to reduce the imaging forces, the SFM experiments were carried out in aqueous environment. For this purpose the wafers were first coated with a monolayer of polymerized Cd-PCA. Then the monolayers, which were to be imaged, were transferred by horizontal dipping onto these substrates. When imaged in the SFM, we generally found that the surface of such films appeared rather structureless at low magnification. This holds for monomeric as well as for polymeric monolayers. At higher magnification, however, ordered patterns appear in both cases. (24) Radmacher, M.; Zimmermann,R. M.; Gaub, H. E. In Amphiphilic membranes; Lipowski, Richter, Eds.; Springer: New York, 1991.
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Langmuir, VoZ.8, No. 7,1992 1773
Figure 8. (a) High-magnification WM image or a monomeric PCA monolayer taken under water. (b) Close-up view of (a).
Figure 9. (a) High-magnification SFM image of a polymeric PCA monolayer taken under water. (b) Close-up view of (a).
Figure 8 shows the surface of the monomeric PCA films at two different magnifications. The surface exhibits a well-pronouncedlattice which extends over all the scanned area. The Fourier transform reveals an oblique lattice with the unit cell of a = b = 4.5 f 1A and g = 70 f 5 O . These values are in good agreement with data from other groups which were obtained by X-rays and electron diffraction on multilayers.20 There are smaller areas in the field,however, where the lattice is less well pronounced. These areas might contain defects, which due to the increased mobility of the neighboring fatty acids prevent high-resolution imaging.24 The lack of resolution might also stem from defects of the substrate. The long wavelength heights variation of several angstroms as they are seen in Figure 8a are not found on the silicon oxide itself but can at low magnifications be seen already on the lower PCA layer when imaged in air. These undulations might stem from insoluble Cd salt clusters which are
trapped between film and substrate. It should also be noted here that, under prolonged scanning, even with weak applied forces, the monomeric film tends to rupture and reorganize into multilayered structures. When polymerized,the PCA films exhibit a much higher durability. Even after hours of scanning, molecular resolution may be achieved from the same spot. Figure 9 shows such a film at two different magnifications. Again a well-pronounced periodic pattern with an amplitude on the order of fractions of angstroms is found. Within the accuracy of our measurements, this lattice is the same as the lattice of the monomeric films. This is not too surprising, as the lattice contraction during polymerization is known to be less than lo%,which is our estimated experimental uncertainty for lateral dimensions. It is also important to note that in films which were polymerized in the low-pressure phase we found no indication for a major disorder in the lattice which again is a good indicator
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1774 Langmuir, Vol. 8, No. 7,1992
vature radius of about 300 A were used, the minimum forcebetween tip and sample is in the range of some nanonewtons. Although a large portion of this force is distributed over a larger area by hydration forces between tip and sample, the remaining point interaction between the last imaging atoms of the tip and the fatty acid molecule or ita hydration shells remains in the order of 10-lo N.24At such forces the molecular order stays preserved only in extended crystals where the cooperativity among the molecules stabilizes the crystalline order. In the vicinity of defects or at crystal boundaries, however, the tip may disturb the packing of the molecules such that even threedimensional reorganizations may occur.
that the above given explanation for the different fluorescence polarization degrees is correct. Despite the fact that layered minerals like mica are much easier to handle as substrates for SFM, we have chosen an amorphous substrate in order to prove unambiguously without interference by the substrate that the SFM may image such films at molecular resolution. The images shown above clearly indicate that the SFM is capable of resolving the lattice of crystals of the fatty acid in both the monomeric and the polymeric form. In a recent study we could show that, under the conditions here, where the samples are sufficiently flat, the image formation is dominated by a localizedinteraction leading to a true point resolution of up to 3 A.26 This means that the images above show individual PCA molecules rather than just the reproduction of their periodic lattice. The fact that both the monomeric and the polymeric films give rise to very similar images in the SFM is very important for the general discussion on the imaging mechanism in SFM. The similarity indicates that the image formation is determined by the surface properties of the lipid film rather than by the properties of the hydrocarbon chain region. If the tip would penetrate into the hydrocarbon chains while imaging, the highly oriented polymer backbone should significantly influence the resolution in one direction. One would in this cme expect to see rows along the direction of the polymer rather than a lattice. This means that the images above show the individual head groups of PCA. The finding that the monomeric films tend to reorganize during extended scanning is easily understandable in view of the considerable forces that act on the molecules while scanning. The forces between tip and sample are determined largely by the tip geometry. In our case, where relatively dull micromanifactured tips with a mean cur-
Acknowledgment. This work was supported by the Deutsche Forschungs-gemeinschaft. We thank Karl Eberle for his skillful technical support and Dominic Benvegnu for carefully reading the paper. We are in debt for helpful discussionswith Helen and Paul Hansma, Helmut Ringsdorf, and Hans Ribi.
(25) Tillmann,R.; Radmacher, M.; Gaub, H. E. Appl. Phys. Lett., in press.
Registry No. PCA, 66990-32-7; PCA (homopolymer),6699033-8.
Concluding Remarks Our study reveals details of the molecular structure of solid monomolecular films of PCA both in the monomeric and in the polymeric state. These results clearly demonstrate the potential of the SFM for the investigations of thin organic films and in turn the potential value of such films for SFM investigations. Such flat and mechanically stable films are ideal candidates for substrates in SFM. Polymeric lipid films of various compositions might be used in future studies to immobilize larger molecules to be imaged by SFM by either chemical bonds or Coulomb forces. Even the design of lateral structures on molecular dimension is conceivable by the use of mixed systems in combination with suitable LB techniques.
