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Observation of chiral domain morphology in a phospholipid Langmuir-Blodgett monolayer by atomic force microscopy
26 September 1994 PHYSICS LETTERS A
ELSEVIER
PhysicsLetters A 193 (1994) 195-198
Observation of chiral domain morphology in a phospholipid Langmuir-Blodgett monolayer by atomic force microscopy Xiao-Min Yang, Dong Xiao, Shou-Jun Xiao, Zu-Hong Lu, Yu Wei National Laboratory of Molecular and Biomolecular Electronics, Southeast University, Nanfing 210018, China
Received 18 January 1994; revised manuscript received 12 July 1994; accepted for publication 15 July 1994 Communicatedby L.J. Sham
Abstract
Using atomic force microscopy, we directly observe monolayer Langmuir-Blodgett (LB) films of dipalmitoylphosphatidylcholine (DPPC) deposited onto a solid substrate. Under specified deposition conditions, we find that the lipid monolayers form two-dimensional chiral morphologies, most with 2-, 3-, 4- or 6-fold rotation symmetry, which is directly related to the enantiomorphic configuration of the lipids composing the monolayer. In addition, the high resolution AFM images also reveal the finestructure of the domains in the two-phase coexistence region as well as within a single solid phase domain, which cannot be identified by optical microscopic techniques.
Monolayers ofphospholipid at the air-water interface have been widely studied [ 1,2 ]. In these studies, fluorescent liquid analogues have been incorporated into these monolayers, permitting epifluorescence observation at the air-water interface[3,4]. However, the use of a fluorescence p r o b e causes some problems. For instance, a high probe concentration may lead to artifacts, while low concentrations require a very sensitive detection system. On the other hand, fluorescence optical microscopy is limited to structures larger than 1/tm in diameter, so it remains difficult to investigate the microstructure of domains and the early stages of domain formation. In addition, little is known about the morphology of phospholipid monolayers after transfer onto solid substrates in the submicrometer regime. Recently atomic force microscopy (AFM) has successfully obtained molecular resolution of different monolayer films transferred to solid substrates [ 5 ],
revealed defects in these films [6] and domain boundaries [7 ], measured friction on them [8 ] as well as the domain structures in phase separated LB films of fatty acids. More recently Mikrut et al. [ 10 ] have reported AFM studies of phospholipid LB films. Here, we report the first AFM observation of the chiral domain morphology in phospholipid monolayers transferred onto solid substrates without any fluorescence probe. The shape of these domains provides direct visual evidence for orientational order in a two-dimensional LB system. Phosphatidylcholines possess a chiral centre at the C-2 carbon atom of the glycerol backbone. Cell membranes contain only one of the two enantiomers, which is the R isomer according to the R / S nomenclature system of Cahn et al. [ 11]. In our experiments, monolayers were composed of either 2R, 3dipalmitoylglycero- 1-phosphorylcholine (R-DPPC, Sigma, 99%), 2S, 3-dipalmitoylglycero-l-phosphor-
0375-9601/94/$07.00 © 1994 ElsevierScience B.V. All fights reserved SSD10375-9601 (94) 00600-8
X.-M. Yang et al. / Physics Letters A 193 (1994) 195-198
196
ylcholine (S-DPPC) or binary mixtures of the R and S isomers without any fluorescent lipid probe. The Langmuir monolayers are prepared on a Face trough. Doubly distilled water was used as the subphase, the temperature of which was controlled to be 18°C with an accuracy of 0.5°C. The surface pressure was measured with a precision of 0.1 m N / m . The DPPC monolayer spread on the water surface was compressed at a controlled rate (Vc) to a prespecifled surface pressure and the monolayer was transferred to hydrophilic mica by vertical dipping with a deposition speed of about 5 m m m i n - 1. We carried out our AFM imaging in the repulsive mode in air with a commercial system (NanoScope III, Digital Instruments, Santa Barbara). Two different scanners were used for surface inspection: a 125 #m scanner for larger areas and a 0.7 ~tm scanner for high resolution. Soft cantilevers were 200/~m long with an integrated pyramidal Si3N 4 tip, with a spring constant of 0.12 N / m . Typical forces for all the measurements were of the order of 10 nN. Fig. 1 shows a pressure-area curve for the DPPC monolayers at the air-water interface. This pressurearea curve is similar to those published [3,4 ], and indicates that under isothermal compression from an expanded state the lipid molecules undergo a fluid to solid phase transition. This phase transition can be detected as the appearance of a lateral phase separation into coexisting domains of fluid and solid lipid. As the DPPC monolayer is compressed the solid phase domains grow at the expense of the fluid lipid. Here, our results are in good agreement with those of
Peters [ 3 ] and McConnell [4] in that solid phase lipid domains first appear on compression at the main phase transition pressure (/rm, see the pressure-area curve in Fig. 1 ) of the order of ~ 4 m N / m , and increase in size and relative area with increasing surface pressure. In our experiments the main phase transition pressure nm is quite reproducible in terms of the pressure-area curve, whereas the absolute value of the pressure is not. Because lipid molecules are sensitive to any small temperature variation, the phase transition point is strongly temperature dependent, which has been studied in our previous report [12]. Fig. 2a gives a typical AFM image of a DPPC monolayer at the main phase transition pressure gm (Fig. 1 ). The bright regions are solid phase lipid and the dark regions represent fluid phase lipid. Comparing the present results with those observed earlier at the air-water interface by fluorescence microscopy [4], we note that domain shapes and sizes are very similar under the same deposition conditions. This provides direct evidence that the crystalline struc-
40-
-•E
30-
e
20-
13_
o o~10" 030
~k
7tm
5b
. 7b
Area
(A2/molecule)
9'0
110
Fig. 1. Surface pressure-area isotherm for the DPPC Langmuir monolayer at 18"C.
Fig. 2. AFM images of DPPC monolayer at the main phase transition point nm (see Fig. 1 ). Scan size 5.0 × 5.0/tm 2, SP = 4 m N / m, Vc= 3.96 A molecule -m min -m.
X.-M. Yang et aL / Physics Letters A 193 (1994) 195-198
tures of the solid phase domains remain unchanged when they are transferred from the air-water interface to solid substrates. In addition, the height measurements show that solid phase lipid regions are about 2.0 nm higher than the fluid phase lipid regions (see Fig. 2b). This indicates that the alkane chains of the solid phase lipids are more closely packed in a nearly perpendicular arrangement than those of the fluid phase lipids on the solid substrate and the fluid phase lipid shows a loose structure. An interesting observation is that, under the specified deposition conditions, the lipid monolayers can form two-dimensional chiral morphologies, most with 4-fold rotation symmetry but sometimes also with 2-, 3-, or 6-fold rotation symmetry. Such handed structures have been observed at the air-water interface [ 13 ], unfortunately, there have been few reports about them on solid substrates[14]. Here, we observe them on solid substrates for the first time. Our observations imply that the chiral properties do not change when phospholipid monolayers are transferred from the air-water interface to the solid substrate. Fig. 3 shows a typical AFM image of chiral structures with 4-fold rotation symmetry, which is composed of R-DPPC. The rotation direction of this spirals is counterclockwise, while Fig. 4 shows chiral solid domains in monolayers composed of S-DPPC. Comparing Fig. 3 with Fig. 4, we easily observe that the rotative direction of the chiral structure is opposite. To understand the chiral nature of the solid phase domains, we also observed monolayers composed of
Fig. 3. Chiral solid phase domains with 4-fold counterclockwise rotation symmetry in monolayers composed of R-DPPC. Scan size 42×42 #m2, SP=5 mN/m, Vc=7.62 A molecule-~ min -~.
197
Fig. 4. Chiral solid phase domains with 4-foldclockwiserotation symmetryin monolayerscomposedof S-DPPC. Scan size 34 X 34 /zm2, SP=5 mN/m, Vc=7.62 A molecule-l min-I.
Fig. 5. Chiral solid phase domains in monolayerscomposedof a racemic mixture without pronouncedchirality. Scan size 32 × 32 #m2, SP=5 mN/m, Vc=7.62 A molecule-~ min-1. a racemic mixture of R-DPPC and S-DPPC under the same deposition conditions as Figs. 3, 4, which are shown in Fig. 5. We observe that these domains have no pronounced chirality compared to domains of RDPPC (Fig. 3) or S-DPPC (Fig. 4). However, careful examination of these domains in the racemic monolayer does show some weak chiral features. Why are chiral solid domains formed in two dimensions? The physical mechanism is now understandable. In principle, the handedness of these solid domains and the sense of the spirals are directly related to the enantiomorphic configuration of the lipids composing the monolayer. A number of our experiments convinced us that the crystal shapes and
198
X.-M. Yang et al. / Physics Letters A 193 (1994) 195-198
sizes o f the solid phase domains depend on a n u m b e r o f factors such as the temperature, subphase composition, and trace impurities in the monolayer system. Moreover, the kinetic factors such as the rate o f compression in crystal growth also have large effects on domain shapes. In our experiments we found that the domain shapes were often round if the phospholipid monolayers were subjected to a slow isothermal compression. This can be attributed to domain formation in the equilibrium state. On the contrary, a high compression speed would produce dendritic domain patterns (data not shown). On the other hand, we also believe these chiral features may involve overlap o f the diffusion fields in the fluid lipid during crystal formation. This diffusion field is long-range in two dimensions. Electrostatic effects may also affect these structures. The solid phase domains formed at the air-water interface must have long-range molecular orientational order. This order is essential for the formation o f chiral domains. A F M images also reveal two fine-structure features in phospholipid monolayers, which cannot be seen by fluorescence optical microscopy due to their small sizes. First, we found a lot of small grains in the fluid phase region, 3 0 - 8 0 n m in size (see fluid phase region of Figs. 4 - 6 ) . We thought these grains were the nuclei of condensed domains. During the process o f solid phase domain growth, these grains failed to grow further due to a n u m b e r o f factors which affect the domain formation. In addition, by increasing the magnification we were able to investigate the fine-
Fig. 6. Fine-structures within a single domain composed of SDPPC. Scan size 10X 10/tm 2, SP=5 mN/m, Vc=7.62 A molecule- ~min- i.
structure within a single domain (shown in Fig. 6). We found that the surface o f the solid phase domain was not homogeneous and some defect structures existed, such as pinholes and impurities (bright spots in the centre o f Fig. 5 ). AFM images revealed these pinholes were about 100-200 n m in diameter and about 1-2 nm in depth. We thought the main reason for these pinholes was that the phospholipid molecules are mobile during two-dimensional crystal formation. However, further experiments will be done to understand these fine-structures in phospholipid monolayer systems. In conclusion, we report the first observation of the chiral domain morphology o f phospholipid monolayers on a solid substrate by AFM, which imply that the chiral properties do not change when phospholipid monolayers are transferred from the air-water interface to a solid substrate. We also found that the shapes and sizes o f the solid phase domains depend on a number o f factors such as temperature, subphase composition, impurities and the rate o f compression. The authors gratefully acknowledge the financial support from the National Natural Science Foundation o f China for this work.
References
[ 1] O. Albrecht, H. Gruler and E. Sackmann, J. Phys. 39 ( 1987) 301. [2] A. Fischer, M. Losche, H. Mohwald and E. Sackmann, J. Phys. Lett. 45 (1984) L785. [ 3 ] R. Peters and K. Beck, Proc. Natl. Acad. Sci. USA 80 (1983) 7183. [4] H.M. McConnell, L.K. Tamm and R.M. Weis, Proc. Natl. Acad. Sci. USA 81 (1984) 3249. [5] D.K. Schwartz et at., Science 257 (1992) 508. [ 6 ] H.G. Hansma et at., Langmuir 7 ( 1991 ) 1051. [ 7 ] J. Garnaes et al., Nature 357 (1992 ) 54. [8 ] E. Meyer et al., Phys. Rev. Lett. 69 (1992) 1777. [9] L.F. Chi et al., Science 259 ( 1993) 213. [ 10] J.M. Mikrut, P. Durra, J.B. Ketterson and R.C. MacDonald, Phys. Rev. B 48 (1993 ) 14479. [ 11 ] H. Hauser et al., Biochim. Biophys. Acta 650 ( 1981 ) 21. [12] J.Y. Fang, Z.H. Lu, L. Wang and Y. Wei, Phys. Lett. A 166 (1992) 373. [ 13 ] R.M. Weis and H.M. McConnell, Nature 310 (1984) 47. [ 14] V.T. Moy, D.J. Keller, H.E. Gaub and H.M. McConnell, J. Phys. Chem. 90 (1986) 3198.
ELSEVIER
PhysicsLetters A 193 (1994) 195-198
Observation of chiral domain morphology in a phospholipid Langmuir-Blodgett monolayer by atomic force microscopy Xiao-Min Yang, Dong Xiao, Shou-Jun Xiao, Zu-Hong Lu, Yu Wei National Laboratory of Molecular and Biomolecular Electronics, Southeast University, Nanfing 210018, China
Received 18 January 1994; revised manuscript received 12 July 1994; accepted for publication 15 July 1994 Communicatedby L.J. Sham
Abstract
Using atomic force microscopy, we directly observe monolayer Langmuir-Blodgett (LB) films of dipalmitoylphosphatidylcholine (DPPC) deposited onto a solid substrate. Under specified deposition conditions, we find that the lipid monolayers form two-dimensional chiral morphologies, most with 2-, 3-, 4- or 6-fold rotation symmetry, which is directly related to the enantiomorphic configuration of the lipids composing the monolayer. In addition, the high resolution AFM images also reveal the finestructure of the domains in the two-phase coexistence region as well as within a single solid phase domain, which cannot be identified by optical microscopic techniques.
Monolayers ofphospholipid at the air-water interface have been widely studied [ 1,2 ]. In these studies, fluorescent liquid analogues have been incorporated into these monolayers, permitting epifluorescence observation at the air-water interface[3,4]. However, the use of a fluorescence p r o b e causes some problems. For instance, a high probe concentration may lead to artifacts, while low concentrations require a very sensitive detection system. On the other hand, fluorescence optical microscopy is limited to structures larger than 1/tm in diameter, so it remains difficult to investigate the microstructure of domains and the early stages of domain formation. In addition, little is known about the morphology of phospholipid monolayers after transfer onto solid substrates in the submicrometer regime. Recently atomic force microscopy (AFM) has successfully obtained molecular resolution of different monolayer films transferred to solid substrates [ 5 ],
revealed defects in these films [6] and domain boundaries [7 ], measured friction on them [8 ] as well as the domain structures in phase separated LB films of fatty acids. More recently Mikrut et al. [ 10 ] have reported AFM studies of phospholipid LB films. Here, we report the first AFM observation of the chiral domain morphology in phospholipid monolayers transferred onto solid substrates without any fluorescence probe. The shape of these domains provides direct visual evidence for orientational order in a two-dimensional LB system. Phosphatidylcholines possess a chiral centre at the C-2 carbon atom of the glycerol backbone. Cell membranes contain only one of the two enantiomers, which is the R isomer according to the R / S nomenclature system of Cahn et al. [ 11]. In our experiments, monolayers were composed of either 2R, 3dipalmitoylglycero- 1-phosphorylcholine (R-DPPC, Sigma, 99%), 2S, 3-dipalmitoylglycero-l-phosphor-
0375-9601/94/$07.00 © 1994 ElsevierScience B.V. All fights reserved SSD10375-9601 (94) 00600-8
X.-M. Yang et al. / Physics Letters A 193 (1994) 195-198
196
ylcholine (S-DPPC) or binary mixtures of the R and S isomers without any fluorescent lipid probe. The Langmuir monolayers are prepared on a Face trough. Doubly distilled water was used as the subphase, the temperature of which was controlled to be 18°C with an accuracy of 0.5°C. The surface pressure was measured with a precision of 0.1 m N / m . The DPPC monolayer spread on the water surface was compressed at a controlled rate (Vc) to a prespecifled surface pressure and the monolayer was transferred to hydrophilic mica by vertical dipping with a deposition speed of about 5 m m m i n - 1. We carried out our AFM imaging in the repulsive mode in air with a commercial system (NanoScope III, Digital Instruments, Santa Barbara). Two different scanners were used for surface inspection: a 125 #m scanner for larger areas and a 0.7 ~tm scanner for high resolution. Soft cantilevers were 200/~m long with an integrated pyramidal Si3N 4 tip, with a spring constant of 0.12 N / m . Typical forces for all the measurements were of the order of 10 nN. Fig. 1 shows a pressure-area curve for the DPPC monolayers at the air-water interface. This pressurearea curve is similar to those published [3,4 ], and indicates that under isothermal compression from an expanded state the lipid molecules undergo a fluid to solid phase transition. This phase transition can be detected as the appearance of a lateral phase separation into coexisting domains of fluid and solid lipid. As the DPPC monolayer is compressed the solid phase domains grow at the expense of the fluid lipid. Here, our results are in good agreement with those of
Peters [ 3 ] and McConnell [4] in that solid phase lipid domains first appear on compression at the main phase transition pressure (/rm, see the pressure-area curve in Fig. 1 ) of the order of ~ 4 m N / m , and increase in size and relative area with increasing surface pressure. In our experiments the main phase transition pressure nm is quite reproducible in terms of the pressure-area curve, whereas the absolute value of the pressure is not. Because lipid molecules are sensitive to any small temperature variation, the phase transition point is strongly temperature dependent, which has been studied in our previous report [12]. Fig. 2a gives a typical AFM image of a DPPC monolayer at the main phase transition pressure gm (Fig. 1 ). The bright regions are solid phase lipid and the dark regions represent fluid phase lipid. Comparing the present results with those observed earlier at the air-water interface by fluorescence microscopy [4], we note that domain shapes and sizes are very similar under the same deposition conditions. This provides direct evidence that the crystalline struc-
40-
-•E
30-
e
20-
13_
o o~10" 030
~k
7tm
5b
. 7b
Area
(A2/molecule)
9'0
110
Fig. 1. Surface pressure-area isotherm for the DPPC Langmuir monolayer at 18"C.
Fig. 2. AFM images of DPPC monolayer at the main phase transition point nm (see Fig. 1 ). Scan size 5.0 × 5.0/tm 2, SP = 4 m N / m, Vc= 3.96 A molecule -m min -m.
X.-M. Yang et aL / Physics Letters A 193 (1994) 195-198
tures of the solid phase domains remain unchanged when they are transferred from the air-water interface to solid substrates. In addition, the height measurements show that solid phase lipid regions are about 2.0 nm higher than the fluid phase lipid regions (see Fig. 2b). This indicates that the alkane chains of the solid phase lipids are more closely packed in a nearly perpendicular arrangement than those of the fluid phase lipids on the solid substrate and the fluid phase lipid shows a loose structure. An interesting observation is that, under the specified deposition conditions, the lipid monolayers can form two-dimensional chiral morphologies, most with 4-fold rotation symmetry but sometimes also with 2-, 3-, or 6-fold rotation symmetry. Such handed structures have been observed at the air-water interface [ 13 ], unfortunately, there have been few reports about them on solid substrates[14]. Here, we observe them on solid substrates for the first time. Our observations imply that the chiral properties do not change when phospholipid monolayers are transferred from the air-water interface to the solid substrate. Fig. 3 shows a typical AFM image of chiral structures with 4-fold rotation symmetry, which is composed of R-DPPC. The rotation direction of this spirals is counterclockwise, while Fig. 4 shows chiral solid domains in monolayers composed of S-DPPC. Comparing Fig. 3 with Fig. 4, we easily observe that the rotative direction of the chiral structure is opposite. To understand the chiral nature of the solid phase domains, we also observed monolayers composed of
Fig. 3. Chiral solid phase domains with 4-fold counterclockwise rotation symmetry in monolayers composed of R-DPPC. Scan size 42×42 #m2, SP=5 mN/m, Vc=7.62 A molecule-~ min -~.
197
Fig. 4. Chiral solid phase domains with 4-foldclockwiserotation symmetryin monolayerscomposedof S-DPPC. Scan size 34 X 34 /zm2, SP=5 mN/m, Vc=7.62 A molecule-l min-I.
Fig. 5. Chiral solid phase domains in monolayerscomposedof a racemic mixture without pronouncedchirality. Scan size 32 × 32 #m2, SP=5 mN/m, Vc=7.62 A molecule-~ min-1. a racemic mixture of R-DPPC and S-DPPC under the same deposition conditions as Figs. 3, 4, which are shown in Fig. 5. We observe that these domains have no pronounced chirality compared to domains of RDPPC (Fig. 3) or S-DPPC (Fig. 4). However, careful examination of these domains in the racemic monolayer does show some weak chiral features. Why are chiral solid domains formed in two dimensions? The physical mechanism is now understandable. In principle, the handedness of these solid domains and the sense of the spirals are directly related to the enantiomorphic configuration of the lipids composing the monolayer. A number of our experiments convinced us that the crystal shapes and
198
X.-M. Yang et al. / Physics Letters A 193 (1994) 195-198
sizes o f the solid phase domains depend on a n u m b e r o f factors such as the temperature, subphase composition, and trace impurities in the monolayer system. Moreover, the kinetic factors such as the rate o f compression in crystal growth also have large effects on domain shapes. In our experiments we found that the domain shapes were often round if the phospholipid monolayers were subjected to a slow isothermal compression. This can be attributed to domain formation in the equilibrium state. On the contrary, a high compression speed would produce dendritic domain patterns (data not shown). On the other hand, we also believe these chiral features may involve overlap o f the diffusion fields in the fluid lipid during crystal formation. This diffusion field is long-range in two dimensions. Electrostatic effects may also affect these structures. The solid phase domains formed at the air-water interface must have long-range molecular orientational order. This order is essential for the formation o f chiral domains. A F M images also reveal two fine-structure features in phospholipid monolayers, which cannot be seen by fluorescence optical microscopy due to their small sizes. First, we found a lot of small grains in the fluid phase region, 3 0 - 8 0 n m in size (see fluid phase region of Figs. 4 - 6 ) . We thought these grains were the nuclei of condensed domains. During the process o f solid phase domain growth, these grains failed to grow further due to a n u m b e r o f factors which affect the domain formation. In addition, by increasing the magnification we were able to investigate the fine-
Fig. 6. Fine-structures within a single domain composed of SDPPC. Scan size 10X 10/tm 2, SP=5 mN/m, Vc=7.62 A molecule- ~min- i.
structure within a single domain (shown in Fig. 6). We found that the surface o f the solid phase domain was not homogeneous and some defect structures existed, such as pinholes and impurities (bright spots in the centre o f Fig. 5 ). AFM images revealed these pinholes were about 100-200 n m in diameter and about 1-2 nm in depth. We thought the main reason for these pinholes was that the phospholipid molecules are mobile during two-dimensional crystal formation. However, further experiments will be done to understand these fine-structures in phospholipid monolayer systems. In conclusion, we report the first observation of the chiral domain morphology o f phospholipid monolayers on a solid substrate by AFM, which imply that the chiral properties do not change when phospholipid monolayers are transferred from the air-water interface to a solid substrate. We also found that the shapes and sizes o f the solid phase domains depend on a number o f factors such as temperature, subphase composition, impurities and the rate o f compression. The authors gratefully acknowledge the financial support from the National Natural Science Foundation o f China for this work.
References
[ 1] O. Albrecht, H. Gruler and E. Sackmann, J. Phys. 39 ( 1987) 301. [2] A. Fischer, M. Losche, H. Mohwald and E. Sackmann, J. Phys. Lett. 45 (1984) L785. [ 3 ] R. Peters and K. Beck, Proc. Natl. Acad. Sci. USA 80 (1983) 7183. [4] H.M. McConnell, L.K. Tamm and R.M. Weis, Proc. Natl. Acad. Sci. USA 81 (1984) 3249. [5] D.K. Schwartz et at., Science 257 (1992) 508. [ 6 ] H.G. Hansma et at., Langmuir 7 ( 1991 ) 1051. [ 7 ] J. Garnaes et al., Nature 357 (1992 ) 54. [8 ] E. Meyer et al., Phys. Rev. Lett. 69 (1992) 1777. [9] L.F. Chi et al., Science 259 ( 1993) 213. [ 10] J.M. Mikrut, P. Durra, J.B. Ketterson and R.C. MacDonald, Phys. Rev. B 48 (1993 ) 14479. [ 11 ] H. Hauser et al., Biochim. Biophys. Acta 650 ( 1981 ) 21. [12] J.Y. Fang, Z.H. Lu, L. Wang and Y. Wei, Phys. Lett. A 166 (1992) 373. [ 13 ] R.M. Weis and H.M. McConnell, Nature 310 (1984) 47. [ 14] V.T. Moy, D.J. Keller, H.E. Gaub and H.M. McConnell, J. Phys. Chem. 90 (1986) 3198.