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Surface potential imaging of phase-separated LB monolayers by scanning Maxwell stress microscopy
Thin Solid Films, 243 (1994) 399-402
399
Surface potential imaging of phase-separated LB monolayers by scanning Maxwell stress microscopy Takahito Inoue and Hiroshi Yokoyama Molecular Physics Section, Electrotechnical Laboratory, 1-1-4 Umezono, Tsukuba-shi, Ibaraki 305 (Japan)
Abstract
It is well known that competing interactions such as electrostatic repulsive and van der Waals attractive forces dominate the dynamics of pattern formation in a variety of structures found in phase-separated Langmuir monolayers. However, the microscopic details of these forces are still largely left unresolved because of the technical limitation on measurements. The scanning Maxwell stress microscope, which we have been developing as a new tool for microscopic observation of electrostatic interactions in organic and biological systems, is a type of scanning probe microscope, designed to image the distribution of the Maxwell stress field over the sample surface. With this technique we have succeeded in obtaining images of the surface potential of a phase-separated Langmuir-Blodgett monolayer of phospholipid with submicron resolution.
I. Introduction
In the last decade, a number of investigations of Langmuir monolayers, mainly employing microfluorescence techniques, have revealed the variety of morphologies of phase separated domains. To explain the apparent stability of these domain shapes, theoretical attempts have been made ascribing the origin of domain shapes to the existence of intermolecular electrostatic interactions [1, 2]. Although the presence of electrostatic interactions has been verified on a macroscopic scale over a few tens of micrometers [3], no microscopic observations, which should provide information on the initial process in the phase separation, have yet been carried out, primarily because no proper experimental technique is available. Scanning force microscopy (SFM) including atomic force microscopy (AFM) allows us to obtain not only surface topography at atomic resolution but also information about physical and chemical properties of surfaces [4, 5]. Particularly in the attractive mode, force microscopy has been used in a number of novel areas ranging from non-contact profiling of surfaces to magnetic [6] and electrostatic imaging [7]. Scanning Maxwell stress microscopy (SMM) is one such technique, designed to image the distribution of the Maxwell stress field over the sample surface [8]. This technique enables precise microscopic measurement of electrostatic interactions in organic and biological systems. An important step towards a better understanding of the details of these processes is expected from observation of surface potential profiles of two-dimensional phospholipid monolayers.
0040-6090/94/$7.00 SSDI 0040-6090(93)04220-M
In this paper, we report investigations on the surface potential profile structure of phase-separated Langmuir-Blodgett (LB) monolayers by SMM. This is a first step towards the understanding of electrostatic interactions between molecules and molecular aggregates in organic and biological systems.
2. Instrumentation
Our SMM system is based on the Nanoscope II A F M (Digital Instruments, Santa Barbara, CA) and is equipped with an external voltage source and a double lock-in amplifier system to detect the oscillating force signals. Cantilevers supplied by Digital Instruments are also used after coating platinum on the silicon nitride pyramidal tip surface to make it electrically conductive. In operation, the conductive tip is held away from the sample surface by a few tens of nanometers, and is laterally scanned, detecting the electrostatic Maxwell stress, instead of the contact repulsive force as in AFM. As the details of the SMM have been described elsewhere [8], only the outline of the principle is given here. The electrostatic Maxwell stress exerts a force field associated with an electromagnetic field. In the case of electric Maxwell stress, with which we concern ourselves exclusively here, it creates a force on a metallic surface ~0(E" E)/2 per unit area, with t0 being the dielectric permittivity of the vacuum and E the electric field. To illustrate the principle of the SMM, let the A F M tip be modeled as a plane conductive surface of area S, separated by distance d from a plane sample surface; then the
© 1994- Elsevier Sequoia. All rights reserved
400
12 lnoue, H. Yokoyama / SurJ~teepotential imaging of LB monolayers
force on the tip held at the electrostatic potential Vt is given by 1 S rt = ~ e0 ~5 ( V s - V,) 2 where Vs denotes the surface potential of the sample surface. If the tip potential is driven by an external voltage source so as to give Vt = Vd.c. + Va.c. sin cot, there appear three Maxwell stress components, i.e. the d.c. component Td.... the fundamental component T~,,, and the second harmonic component T2,o. As readily verified, the surface potential Vs can be obtained from the co-component. The tip-sample separation can be obtained from the 2co-component and is controlled by means of a z-piezo actuator in such a way as to fix the T2~,, at a constant value. Since the 2co-component is proportional to ~o/d 2, SMM can simultaneously image the surface potential and eo/d 2, the latter of which gives the topography, when the sample is electrically conductive. Surface potential measurements have traditionally been performed with the Kelvin method which employs a vibrating planar electrode [9]. In the Kelvin method the lateral resolution lies usually in the millimeter to centimeter range and is primarily limited by the dimension of the Kelvin electrode. For the purpose of microscopic surface potential measurements, a micro Kelvin technique has been developed in which a thin needle electrode is utilized [10, 11]. The sensitivity of this technique, however, depends on the electrode size, since a reduction of electrode size leads directly to a reduction of the induced current. Consequently, the micro Kelvin method becomes more and more disadvantageous as the electrode is miniaturized. The SMM, in contrast, detects the Maxwell stress which stays constant irrespective of the electrode size, as long as the applied voltage is held constant [8], making the SMM more suitable for microscopic observation.
3. Experimental details Monolayers were spread on water from a mixed solution of dipalmitoylphosphatidylcholine (DPPC) including 2 mol% of the fluorescent lipid probe N-(7nitrobenz-2-oxa- 1,3-diazol-4-yl)- 1,2-dihexadecanoylsn-glycero-3-phosphoethanolamine (NBD-PE) in a chloroform solution. The phospholipids used for the experiments were obtained with a degree of purity higher than 99%. The phospholipid probes were used without further purification. Monolayers were formed at the air-water interface of a home-made rectangular Teflon trough (285 x 260mm). The surface pressure was measured with a Wilhelmy balance. The monolayer films were rested for 10min before compression to
allow sufficient solvent removal and then compressed by a movable Teflon barrier at a constant rate of 0.1 mm s -~. The monolayers were transferred by the LB technique onto a vacuum deposited aluminum film on a glass plate at room temperature. The LB monolayer was observed through an Olympus IMT-2 fluorescence microscope and the behavior of monolayers at the air-water interface during compression was observed by using a long working distance objective on this fluorescence microscope equipped with a Teflon trough and a movable barrier driven by a motorized actuator. The fluorescent image, captured by a silicon intensifier target (SIT) video camera attached to the microscope, was recorded on a video cassette recorder.
4. Results and discussion In the last decade microfluorescence studies of the phospholipid monolayer have shown that the monolayer undergoes a fluid to solid phase transition under isothermal compression from the expanded state. This transition is marked by a phase separation into coexisting fluid- and solid-phase lipid domains. Although the details of this mechanism are not clear, it has been conjectured that this process is dominated by longrange coulombic interactions between molecules and domains in the monolayer. In combination with a microfluorescence technique, SMM serves as a powerful technique to study surface potential profiles of two dimensional phospholipid monolayer. A vacuum deposited aluminum film on a glass plate was used as a substrate for the LB technique. At first, we confirmed that the surface potential showed no fluctuation over the surface with the potential difference of about 1 V between platinum (tip) and aluminum (substrate), as expected from work function values of these materials [ 12]. Figures l(a) and l(b) show fluorescence micrographs of DPPC monolayers containing the fluorescent lipid probe NBD-PE deposited by the LB technique at a surface pressure of 10 mN m-~ respectively. The surface pressure-area isotherm of the DPPC mixture was observed to have a conspicuous kink at about 10 m N m followed by a plateau, indicating the occurrence of the so-called liquid expanded ( L E ) - l i q u i d condensed (LC) transition [ 13]. At 5 mN m I, before this L E - L C transition takes place, the fluorescence microscopy showed a uniform bright image without microscopic structures as shown in Figs. l(a) and l(b). In the monolayer transferred at 10 mN m ~ (Fig. l(a)), we observed the development of small circular regions, which may be identified as the LC domains, inside the LE background. The size of the LC regions is apparently a few
T. Inoue, H. Yokoyama / Surface potential imaging of LB monolayers
401
Fig. 2. Surface potential images of DPPC monolayers containing NBD-PE deposited at a surface pressure of (a) 10 mN m t and (b) 40mNm i. Scan size is 10x 10~tm2. Fig. 1. Fluorescence micrographs of DPPC monolayers containing NBD-PE deposited by the LB technique at a surface pressure of (a) 10 mN m i and (b) 40 mN m ~. The bar indicates 50 ~tm. Brightness and contrast in (a) and (b) were optimized to enhance visibility, so that intensity differences between (a) and (b) have no definite meaning. micrometers in diameter and the distribution of these bright patches is fairly uniform. We also observed a small number of dark regions, which are presumably in the solid condensed (SC) phase. The size of the SC regions grew with increasing surface pressure and reached 10-20 ~tm diameter at the surface pressure of 40 m N m - 1 as shown in Fig. 1(b). Figures 2(a) and 2(b) show surface potential images (10 x 10 ~tm2 scan size) of D P P C monolayers containing N B D - P E deposited at a surface pressure of 10 m N m - ~ and 40 m N m - 1 respectively, with the same preparations as shown in Fig. 1. Corresponding to the small LC domains in Fig. l(a), we see in Fig. 2(a) circular regions 0.5-21am in diameter, marked by higher surface potentials. The actual surface potential difference between LC (the circular domain) and LE (the surrounding) region was between 5 0 m V and
100mV. With the increase in surface pressure, the surface potential of the resultant D P P C LB film was observed to rise, reaching at 40 m N m -1 about 2 0 0 300 mV higher than the mean value for the coexistence region. As shown in Fig. 2(b) the LC domains were concomitantly largely smeared, although a residue of the circular domains still clearly remained. An intensively investigated by Keller et al. [2], the shape of the coexistence L E / L C domains are thought to be determined by the competition between the electrostatic repulsive interaction, which favors elongated domains, and the van der Waals interaction, preferring circular domains. Consequently, if the electrostatic interactions are to play an essential role in the L E - L C transition, other than shaping macroscopic domains, we can intuitively expect to observe characteristically distorted domain shapes even for critical nuclei. The S M M observation of the LC domains shown in Fig. 2(a) demonstrates unequivocally the presence of electrostatic interactions in the phase separated monolayer; however, on the basis of the above argument, the good circular shapes of submicron LC domains suggest that
402
T. Inoue, H. Yokoyama / Surface potential imaging of LB monolayers
the electrostatic interaction is not a primary driving force of the transition. According to the recent theoretical study by Andelman et al. [1], the spatial period of alternating solid and fluid stripes associated with a second-order phase transition in Langmuir monolayers is of the order of 100 nm. Despite this prediction, no microscopic measurement of surface potential in related systems has yet been carried out. Although the present SMM study has shown the surface potential profile on the LB monolayers on the relevant spatial scale, complete understanding of the phenomenon entials direct observations ofmonolayers at the air-water interface. The development of a new SMM that is applicable to water surface is now in progress. References 1 D. Andelman, F. Brochard and J. F. Joanny, J. Chem. Phys., 86 (1987) 3673.
2 D. J. Keller, H. M. McConnell and V. T. Moy, J. Phys. Chem., 90 (1986) 2311. 3 W. M. Heckl, H. Baumg~irtner and H. M6hwald, Thin Solid Films, 173 (1989) 269. 4 R. J. Behm, N. Garcia and H. Rohrer (eds.), Scanning Tunneling Microscopy and Related Methods, Kluwer, Dordrecht, 1990. 5 R. Wiesendanger and H.-J. Giintherodt (eds.), Scanning Tunneling Microscopy, Vol. 2, Springer, Berlin, 1992. 6 Y. Martin and H. K. Wickramasinghe, Appl. Phys. Lett., 50 (1987) 1455. 7 M. Nonnenmacher, M. P. O'Boyle and H. K. Wickramasinghe, Appl. Phys. Left., 58 (1991) 2921. 8 H. Yokoyama and T. lnoue, Thin Solid Films, in press. 9 W. A. Zisman, Rev. Sci. Instrum., 3 (1932) 367. 10 H. Baumg~rtner and H. D. Liess, Rev. Sci. Instrum., 59 (1988) 802. 11 R. M~ickel, H. Baumg5rtner and J. Ren, Rev. Sci. Instrum., 64 (1993) 694. 12 Handbook of Chemistry and Physics, 63rd edn, CRC Press, Boca Raton, FL, 1982, E78-79. 13 O. Albrecht, H. Gruler and E. Sackmann, J. Phys. (Paris), 39 (1978) 301.
399
Surface potential imaging of phase-separated LB monolayers by scanning Maxwell stress microscopy Takahito Inoue and Hiroshi Yokoyama Molecular Physics Section, Electrotechnical Laboratory, 1-1-4 Umezono, Tsukuba-shi, Ibaraki 305 (Japan)
Abstract
It is well known that competing interactions such as electrostatic repulsive and van der Waals attractive forces dominate the dynamics of pattern formation in a variety of structures found in phase-separated Langmuir monolayers. However, the microscopic details of these forces are still largely left unresolved because of the technical limitation on measurements. The scanning Maxwell stress microscope, which we have been developing as a new tool for microscopic observation of electrostatic interactions in organic and biological systems, is a type of scanning probe microscope, designed to image the distribution of the Maxwell stress field over the sample surface. With this technique we have succeeded in obtaining images of the surface potential of a phase-separated Langmuir-Blodgett monolayer of phospholipid with submicron resolution.
I. Introduction
In the last decade, a number of investigations of Langmuir monolayers, mainly employing microfluorescence techniques, have revealed the variety of morphologies of phase separated domains. To explain the apparent stability of these domain shapes, theoretical attempts have been made ascribing the origin of domain shapes to the existence of intermolecular electrostatic interactions [1, 2]. Although the presence of electrostatic interactions has been verified on a macroscopic scale over a few tens of micrometers [3], no microscopic observations, which should provide information on the initial process in the phase separation, have yet been carried out, primarily because no proper experimental technique is available. Scanning force microscopy (SFM) including atomic force microscopy (AFM) allows us to obtain not only surface topography at atomic resolution but also information about physical and chemical properties of surfaces [4, 5]. Particularly in the attractive mode, force microscopy has been used in a number of novel areas ranging from non-contact profiling of surfaces to magnetic [6] and electrostatic imaging [7]. Scanning Maxwell stress microscopy (SMM) is one such technique, designed to image the distribution of the Maxwell stress field over the sample surface [8]. This technique enables precise microscopic measurement of electrostatic interactions in organic and biological systems. An important step towards a better understanding of the details of these processes is expected from observation of surface potential profiles of two-dimensional phospholipid monolayers.
0040-6090/94/$7.00 SSDI 0040-6090(93)04220-M
In this paper, we report investigations on the surface potential profile structure of phase-separated Langmuir-Blodgett (LB) monolayers by SMM. This is a first step towards the understanding of electrostatic interactions between molecules and molecular aggregates in organic and biological systems.
2. Instrumentation
Our SMM system is based on the Nanoscope II A F M (Digital Instruments, Santa Barbara, CA) and is equipped with an external voltage source and a double lock-in amplifier system to detect the oscillating force signals. Cantilevers supplied by Digital Instruments are also used after coating platinum on the silicon nitride pyramidal tip surface to make it electrically conductive. In operation, the conductive tip is held away from the sample surface by a few tens of nanometers, and is laterally scanned, detecting the electrostatic Maxwell stress, instead of the contact repulsive force as in AFM. As the details of the SMM have been described elsewhere [8], only the outline of the principle is given here. The electrostatic Maxwell stress exerts a force field associated with an electromagnetic field. In the case of electric Maxwell stress, with which we concern ourselves exclusively here, it creates a force on a metallic surface ~0(E" E)/2 per unit area, with t0 being the dielectric permittivity of the vacuum and E the electric field. To illustrate the principle of the SMM, let the A F M tip be modeled as a plane conductive surface of area S, separated by distance d from a plane sample surface; then the
© 1994- Elsevier Sequoia. All rights reserved
400
12 lnoue, H. Yokoyama / SurJ~teepotential imaging of LB monolayers
force on the tip held at the electrostatic potential Vt is given by 1 S rt = ~ e0 ~5 ( V s - V,) 2 where Vs denotes the surface potential of the sample surface. If the tip potential is driven by an external voltage source so as to give Vt = Vd.c. + Va.c. sin cot, there appear three Maxwell stress components, i.e. the d.c. component Td.... the fundamental component T~,,, and the second harmonic component T2,o. As readily verified, the surface potential Vs can be obtained from the co-component. The tip-sample separation can be obtained from the 2co-component and is controlled by means of a z-piezo actuator in such a way as to fix the T2~,, at a constant value. Since the 2co-component is proportional to ~o/d 2, SMM can simultaneously image the surface potential and eo/d 2, the latter of which gives the topography, when the sample is electrically conductive. Surface potential measurements have traditionally been performed with the Kelvin method which employs a vibrating planar electrode [9]. In the Kelvin method the lateral resolution lies usually in the millimeter to centimeter range and is primarily limited by the dimension of the Kelvin electrode. For the purpose of microscopic surface potential measurements, a micro Kelvin technique has been developed in which a thin needle electrode is utilized [10, 11]. The sensitivity of this technique, however, depends on the electrode size, since a reduction of electrode size leads directly to a reduction of the induced current. Consequently, the micro Kelvin method becomes more and more disadvantageous as the electrode is miniaturized. The SMM, in contrast, detects the Maxwell stress which stays constant irrespective of the electrode size, as long as the applied voltage is held constant [8], making the SMM more suitable for microscopic observation.
3. Experimental details Monolayers were spread on water from a mixed solution of dipalmitoylphosphatidylcholine (DPPC) including 2 mol% of the fluorescent lipid probe N-(7nitrobenz-2-oxa- 1,3-diazol-4-yl)- 1,2-dihexadecanoylsn-glycero-3-phosphoethanolamine (NBD-PE) in a chloroform solution. The phospholipids used for the experiments were obtained with a degree of purity higher than 99%. The phospholipid probes were used without further purification. Monolayers were formed at the air-water interface of a home-made rectangular Teflon trough (285 x 260mm). The surface pressure was measured with a Wilhelmy balance. The monolayer films were rested for 10min before compression to
allow sufficient solvent removal and then compressed by a movable Teflon barrier at a constant rate of 0.1 mm s -~. The monolayers were transferred by the LB technique onto a vacuum deposited aluminum film on a glass plate at room temperature. The LB monolayer was observed through an Olympus IMT-2 fluorescence microscope and the behavior of monolayers at the air-water interface during compression was observed by using a long working distance objective on this fluorescence microscope equipped with a Teflon trough and a movable barrier driven by a motorized actuator. The fluorescent image, captured by a silicon intensifier target (SIT) video camera attached to the microscope, was recorded on a video cassette recorder.
4. Results and discussion In the last decade microfluorescence studies of the phospholipid monolayer have shown that the monolayer undergoes a fluid to solid phase transition under isothermal compression from the expanded state. This transition is marked by a phase separation into coexisting fluid- and solid-phase lipid domains. Although the details of this mechanism are not clear, it has been conjectured that this process is dominated by longrange coulombic interactions between molecules and domains in the monolayer. In combination with a microfluorescence technique, SMM serves as a powerful technique to study surface potential profiles of two dimensional phospholipid monolayer. A vacuum deposited aluminum film on a glass plate was used as a substrate for the LB technique. At first, we confirmed that the surface potential showed no fluctuation over the surface with the potential difference of about 1 V between platinum (tip) and aluminum (substrate), as expected from work function values of these materials [ 12]. Figures l(a) and l(b) show fluorescence micrographs of DPPC monolayers containing the fluorescent lipid probe NBD-PE deposited by the LB technique at a surface pressure of 10 mN m-~ respectively. The surface pressure-area isotherm of the DPPC mixture was observed to have a conspicuous kink at about 10 m N m followed by a plateau, indicating the occurrence of the so-called liquid expanded ( L E ) - l i q u i d condensed (LC) transition [ 13]. At 5 mN m I, before this L E - L C transition takes place, the fluorescence microscopy showed a uniform bright image without microscopic structures as shown in Figs. l(a) and l(b). In the monolayer transferred at 10 mN m ~ (Fig. l(a)), we observed the development of small circular regions, which may be identified as the LC domains, inside the LE background. The size of the LC regions is apparently a few
T. Inoue, H. Yokoyama / Surface potential imaging of LB monolayers
401
Fig. 2. Surface potential images of DPPC monolayers containing NBD-PE deposited at a surface pressure of (a) 10 mN m t and (b) 40mNm i. Scan size is 10x 10~tm2. Fig. 1. Fluorescence micrographs of DPPC monolayers containing NBD-PE deposited by the LB technique at a surface pressure of (a) 10 mN m i and (b) 40 mN m ~. The bar indicates 50 ~tm. Brightness and contrast in (a) and (b) were optimized to enhance visibility, so that intensity differences between (a) and (b) have no definite meaning. micrometers in diameter and the distribution of these bright patches is fairly uniform. We also observed a small number of dark regions, which are presumably in the solid condensed (SC) phase. The size of the SC regions grew with increasing surface pressure and reached 10-20 ~tm diameter at the surface pressure of 40 m N m - 1 as shown in Fig. 1(b). Figures 2(a) and 2(b) show surface potential images (10 x 10 ~tm2 scan size) of D P P C monolayers containing N B D - P E deposited at a surface pressure of 10 m N m - ~ and 40 m N m - 1 respectively, with the same preparations as shown in Fig. 1. Corresponding to the small LC domains in Fig. l(a), we see in Fig. 2(a) circular regions 0.5-21am in diameter, marked by higher surface potentials. The actual surface potential difference between LC (the circular domain) and LE (the surrounding) region was between 5 0 m V and
100mV. With the increase in surface pressure, the surface potential of the resultant D P P C LB film was observed to rise, reaching at 40 m N m -1 about 2 0 0 300 mV higher than the mean value for the coexistence region. As shown in Fig. 2(b) the LC domains were concomitantly largely smeared, although a residue of the circular domains still clearly remained. An intensively investigated by Keller et al. [2], the shape of the coexistence L E / L C domains are thought to be determined by the competition between the electrostatic repulsive interaction, which favors elongated domains, and the van der Waals interaction, preferring circular domains. Consequently, if the electrostatic interactions are to play an essential role in the L E - L C transition, other than shaping macroscopic domains, we can intuitively expect to observe characteristically distorted domain shapes even for critical nuclei. The S M M observation of the LC domains shown in Fig. 2(a) demonstrates unequivocally the presence of electrostatic interactions in the phase separated monolayer; however, on the basis of the above argument, the good circular shapes of submicron LC domains suggest that
402
T. Inoue, H. Yokoyama / Surface potential imaging of LB monolayers
the electrostatic interaction is not a primary driving force of the transition. According to the recent theoretical study by Andelman et al. [1], the spatial period of alternating solid and fluid stripes associated with a second-order phase transition in Langmuir monolayers is of the order of 100 nm. Despite this prediction, no microscopic measurement of surface potential in related systems has yet been carried out. Although the present SMM study has shown the surface potential profile on the LB monolayers on the relevant spatial scale, complete understanding of the phenomenon entials direct observations ofmonolayers at the air-water interface. The development of a new SMM that is applicable to water surface is now in progress. References 1 D. Andelman, F. Brochard and J. F. Joanny, J. Chem. Phys., 86 (1987) 3673.
2 D. J. Keller, H. M. McConnell and V. T. Moy, J. Phys. Chem., 90 (1986) 2311. 3 W. M. Heckl, H. Baumg~irtner and H. M6hwald, Thin Solid Films, 173 (1989) 269. 4 R. J. Behm, N. Garcia and H. Rohrer (eds.), Scanning Tunneling Microscopy and Related Methods, Kluwer, Dordrecht, 1990. 5 R. Wiesendanger and H.-J. Giintherodt (eds.), Scanning Tunneling Microscopy, Vol. 2, Springer, Berlin, 1992. 6 Y. Martin and H. K. Wickramasinghe, Appl. Phys. Lett., 50 (1987) 1455. 7 M. Nonnenmacher, M. P. O'Boyle and H. K. Wickramasinghe, Appl. Phys. Left., 58 (1991) 2921. 8 H. Yokoyama and T. lnoue, Thin Solid Films, in press. 9 W. A. Zisman, Rev. Sci. Instrum., 3 (1932) 367. 10 H. Baumg~rtner and H. D. Liess, Rev. Sci. Instrum., 59 (1988) 802. 11 R. M~ickel, H. Baumg5rtner and J. Ren, Rev. Sci. Instrum., 64 (1993) 694. 12 Handbook of Chemistry and Physics, 63rd edn, CRC Press, Boca Raton, FL, 1982, E78-79. 13 O. Albrecht, H. Gruler and E. Sackmann, J. Phys. (Paris), 39 (1978) 301.