Microstructure of thin Langmuir-Blodgett films of dipalmitoylphosphatidylcholine: electron microscopic images replicated with plasma polymerized film by glow discharge

Chemistry and Physics of

LIPIDS ELSEVIER SCIENTIFIC ptrB{ ISHERS IRELAND

Chemistry and Physics of Lipids 66 (1993) 219-223

Short communication

Microstructure of thin Langmuir-Blodgett films of dipalmitoylphosphatidylcholine: electron microscopic images replicated with plasma polymerized film by glow discharge Emiko Okamura

~a

a

, Junzo U m e m u r a , Keiji Iriyama b, Toshinari Araki c

alnstitute for Chemical Research, Kyoto Universiry, Uji, Kyoto-fu 611, Japan hlnstitute of Medical Science, Jikei University School of Medicine, Nishi-shinbashi. Minato-ku, Tokyo 105, Japan CFrontier Technology Research Institute, Tokyo Gas Company Ltd, Shibaura. Minato-ku, Tokyo 105, Japan

(Received 11 September 1992; revision received 26 January 1993; accepted 8 February 1993)

Abstract

Electron-microscopic images of 1- and 5-monolayer LB films of dipalmitoylphosphatidylcholine, transferred to a glass substrate at various surface pressures encompassing the plateau region of the surface pressure-area isotherm, were observed by using a plasma polymerization replica method. One-monolayer films are flat and there is no substantial evidence of structural defects up to the high surface pressures. Crystalline domain structures appear in the 5-monolayer films even at the low surface pressures below the plateau, which accords well with our previous spectroscopic results. The domains decrease their size with increases in surface pressure and finally disappear at the high surface pressures above the plateau. These are explained by the formation of a closely packed homogeneous structure around the plateau region. Further compression of the monolayer before the collapse pressure leads to the reappearance of the inhomogeneous crystalline domain structures, due to the local collapse of the monolayer just before the main collapse pressure. Key words." Dipalmitoylphosphatidylcholine; Langmuir-Blodgett film; Electron microscopy; Microstructure; Plasma polymerization replica film

1. Introduction

The microstructure o f Langmuir-Blodgett (LB) films is important as one o f the d o m i n a n t factors * Corresponding author.

characterizing film properties. The structure o f LB films o f L-a-dipalmitoylphosphatidylcholine (DPPC) has been o f great interest to us, since the surface pressure (r)-area (A) isotherm o f a D P P C monolayer shows a clear plateau region which is ascribed to the liquid-expanded (LE)/liquid-

0009-3084/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0009-3084(93)02161-J

E, Okamura et al. / Chem, Phys. Lipids 66 (1993) 219-223

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condensed (LC) phase transition. Using highsensitivity FTIR and Raman spectroscopy, we previously investigated surface-pressure dependence of the molecular structure and orientation in thin LB films of DPPC [1,2] and found that (i) in the LC phase, the hydrocarbon chains of DPPC were of all-trans conformation and highly oriented at the tilting angle of -~20° from the surface normal, (ii) in the LE phase, the chain segments were loosely packed and almost randomly oriented with a large number of gauche conformers and (iii) the LE/LC transition corresponded to a reorientation process of the hydrocarbon chains from an almost randomly oriented state to a highly ordered one with the increase in the number of all-trans conformers. We also pointed out that the loosely packed hydrocarbon chains in the LB films deposited at low surface pressures were gradually reoriented with time and finally formed stable and highly ordered aggregates similar to those in the LC phase. Recently Iriyama et al. [3-8] have proposed a plasma polymerization replica method for transmission electron microscopic measurements, in order to visualize the film surface structure on the molecular scale. This method seems to be a powerful tool for the microstructural analysis of extremely small and ultra-thin samples such as LB films and biomaterials without introduction of any significant artifact like staining and shadowing [3,4,9]. So far, they have succeeded in visualizing the microstructure of monolayer LB films of several amphlphilic dyes, fatty acids and phospholipid L-ct-dioleoylphosphatidylcholine (DOPC) [3-6,10]. In order to visualize and confirm the pressureinduced and time-dependent structural difference in DPPC LB films reported on the basis of our spectroscopic measurements [1,2] we applied the plasma polymerization replica method to the study of 1- and 5-monolayer LB films of DPPC deposited at various surface pressures encompassing the plateau region of the lr-A isotherm (Fig. 1), and discussed the film thickness and surface pressure dependences upon the microstructures of the LB films. In particular, we succeeded in visualizing stable crystalline domain structures in the multilayer LB films at low surface pressures, which supported our previous spectroscopic results [2].

2. Experimental procedures The LB films of DPPC were prepared by our newly developed method [1,2]. Briefly, DPPC monolayers were spread from a 1.4 x 10-3 M chloroform solution on the pure water surface at 20°C. The monolayers were transferred onto a glass substrate at surface pressures of (a) 1.5, (b) 5.5, (c) 30 and (d) 45 mN m -1, the points (a) and (b) being below and around the plateau pressure, respectively, and (c) and (d) above it (Fig. 1). The first monolayer was prepared by the vertical dipping (or LB) method, and the succeeding four monolayers were deposited by the horizontal lifting method. Transfer ratios were found to be 1.1 + 0.1 for the first monolayer and 1.9 + 0.1 for the succeeding monolayers at all the surface pressures examined. The glass substrates covered

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0.6

0.8

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Fig. 1. Surface pressure-area isotherm of a DPPC monolayer on water at 20°C and typical replica images of a monolayer transferred to a glass substrate at (a) 1.5, (b) 5.5, (c) 30 and (d) 45 mN m -I.

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by the LB films of DPPC were replicated with plasma polymerized film by glow discharge according to the same procedure described elsewhere [3-7,9]. Each replica film was subjected to electron microscopic measurements by a JEOL model JEM 100 S. 3. Results

Typical replica images of the l-monolayer DPPC LB films prepared at various surface pressures are demonstrated in Fig. 1. Apparently, images of the films deposited at the points (a), (b) and (c) in the ~r-A isotherm are flat on the electron microscopic scale with the resolution about 2 nm [4]. The images also resemble those of 1-monolayer LB films of L-c~-dioleoylphosphatidylcholine (DOPC) deposited on a glass substrate at 8 and 20 mN m -1 (the LE phase) [10]. These facts mean that the LB films of DPPC are of high quality up to the high surface pressures above the plateau (the LC phase). On the other hand, small inhomogeneity in microstructure is observed at high surface pressure of 45 mN m -I (Fig. ld). This may be due to the local collapse occurring in the monolayer during the compression at high surface pressures before the main collapse, as already pointed out from the electron microscopic studies on monolayers of several fatty acids [6,11] and DPPC [12]. Here it should be noted that the image at the LE/LC transition region (Fig. l b) does not exhibit any domain structures. The reason will be discussed later in detail. The replica images of the 5-monolayer DPPC LB films are evidently different from those of the l-monolayer films mentioned above. As shown in Fig. 2a, crystal-like domain structures are widely distributed even at a low surface pressure of 1.5 mN m -l (the LE phase). This pattern supports the appearance of stable and highly ordered aggregates even at the low surface pressures, which is caused by the gradual crystallization of the loosely packed and disordered molecules observed immediately after the film preparation [2]. The crystalline domain structures are also found for the films prepared at 5.5 mN m -~ around the plateau (Fig. 2b). In this case, however, the domain size is smaller than that at 1.5 mN m -l. Further, the domain structures almost disappear at the

high surface pressure of 30 mN m -l above the plateau (Fig. 2c). Iriyama et al. have already reported that the particle-like structures observed for the monolayer LB films of DOPC prepared at - 0 . 0 mN m -J (the area of 1.5 nm 2 molecule -1) diminish at higher surface pressures [10]. From displacement current measurements of a DOPC monolayer spread on the water surface, they have also demonstrated that the DOPC molecules are reoriented at - 1 . 5 nm 2 molecule -1, since a large displacement current is generated along with the change in the direction of molecular dipole moment. The present result of the disappearance of the domain structure in the DPPC LB films at 30 mN m -l quite resembles these electron microscopic findings of the DOPC films. It is therefore considered that the DPPC molecules are reoriented around the plateau region. Further, the decrease in the domain size with increases in the surface pressure shown in Fig. 2a-c can be interpreted as the decrease in gaps between the domains. As pointed out previously [1,2], there are large numbers of highly oriented DPPC molecules above the plateau with the hydrocarbon chains of all trans conformations. These molecules can be related to the increase in the number of nuclei upon crystallization, and cause the domain-size decrease. The domain structure reappears in the 5monolayer LB films of DPPC prepared at the high surface pressure of 45 mN m -l, as shown in Fig. 2d. In this case, the domain size is much larger than that at low surface pressures shown in Fig. 2a,b. In addition, the crystal-like domains also grow in a piled-up fashion. The reappearance of the inhomogeneous structure at high surface pressures is due to the local collapse during the compression before the main collapse pressure, and also originates from the small inhomogeneity in microstructures observed for 1-monolayer films at 45 mN m -1 mentioned before (Fig. ld). 4. Discussion

The present results on replica images of DPPC LB films clearly visualize the difference in surface microstructure of the films depending on the film thickness and the surface pressure. At first the images of 1-monolayer LB films are flat and there is

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no evidence of the existence of domain structures. The flat microscopic image of the monolayer films in LE and LC phases (Fig. la,c) agrees well with previous investigations based on the electron microscopic observation of monomolecular LB films

of DPPC [13,14]. On the other hand, the homogeneous and flat image observed at the LE/LC transition region (Fig. lb) contradicts the existence of domain structures already reported by many other investigations on fluorescence micros-

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40Ohm

400nm

Fig. 2. The replica images of 5-monolayer LB films of DPPC deposited on a glass substrate at (a) 1.5, (b) 5.5, (c) 30 and (d) 4 5 m N m -I.

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copy [15-18] and brewster angle microscopy [19] o f the monolayers at the air-water interface. The most probable reason is that the domain size with diameters - 5 - 2 0 t~m [16,19] is so large that we unfortunately could not visualize any domain shapes but only detect the inner part o f the domain structures on the electron microscopic scale in this study. This may be reasonable, since it is considered that the crystalline domain structures are largely distributed compared with the a m o r p h o u s liquid-crystalline phase at the pressure near the end o f the plateau region (see the 7r-A isotherm in Fig. 1). However, p o o r resolution o f domain structures is still possible because o f the relatively small value o f the domain height (0.7-1.1 nm) [14], although the difference in height ( - 0.3 nm) could already be resolved for m o n o m o l e c u l a r LB films o f stearic acid by using a similar method [8]. The second point that should be emphasized is that 5-monolayer LB films show crystalline domain structures even at low surface pressures (the LE phase), as demonstrated in Fig. 2a. One o f the plausible reasons is the structural perturbation during the transfer process. However, it is not the case, because our F T I R spectroscopic data concerning pressure-dependent frequency shift in the multilayer LB films [1] are in g o o d agreement with those for monolayers at the air-water interface [20]. Further, there is no possibility o f imperfect deposition, since the transfer ratios obtained are almost equal to unity per monolayer. Finally, we interpret the difference o f the images between 1- and 5-monolayer films at low surface pressures (the LE phase) as follows. In 1monolayer LB films, the crystalline domain structures are not formed, presumably because the crystallization is prevented by the adhesive interaction between the monolayer and the solid substrate. In 5-monolayer films, the molecules are at first homogeneously deposited. N o crystallization occurs at this stage and the molecules are in a disordered and loosely packed state. However, the molecules tend to self-aggregate, as is deduced from the extremely low critical micelle concentration (4.6 × 10 -l° M) [21]. Therefore, the molecules are slowly reoriented and crystallized at the next stage, to finally form stable domain structures. This process is valid,

since there is a g o o d correspondence with the reorientation process o f the multilayer LB films prepared at low surface pressures obtained from our R a m a n spectroscopic data [2].

5. Acknowledgements The authors are grateful to Prof. T. T a k e n a k a o f K y o t o University for critical reading o f the manuscript and Dr. M. M a t s u m o t o o f K y o t o University for valuable discussions on the electron micrographs.

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