Langmuir 1994,10, 2081-2083
2081
Cryo-TransmissionElectron Microscopy of Amphiphilic Monolayers of C 3 1 h O H Deposited on Holey Grids J. Majewski and L.Margulis* Department of Materials and Interfaces, The Weizmann Institute of Science, Rehovot 76100, Israel Received March 14, 1994. In Final Form: May 17, 1994@ Monolayers of the long-chain alcohol C31H630H were spread over a water surface and deposited onto plastic holey grids. The water substrate was then vitrified by fast freezing in liquid ethane, and the samples obtained were studied by cryo-Transmission electronmicroscopy. Electron images and diffraction patterns, indicative of two-dimensional crystalline self-assembly,are discussed. Morphological features of the monolayer films were also obtained. The method should be of importance for the study of the structure of membranes, Langmuir-Blodgett films,and nucleation processes oftwo- and three-dimensional crystals.
Introduction Self-assembling of amphiphilic molecules into ordered two-dimensional (2-D) domains on liquid surfaces plays an impoitant role in many fields of pure and applied science.l+ This phenomenon controls the properties of Langmuir-Blodgett films as well as crystal growth induced by a m o n ~ l a y e r . ~ ~It~ plays ~ ~ - lalso l a substantial role in molecular electronics, construction of molecular sensors, etc. The self-assemblyappears to be a widespread phenomenon. Several methods were used to investigate the structural and morphological properties of such 2-D assemblies: Brewster angle m i ~ r o s c o p y , fluorescence ~~J~ micro~copy,l~-'~ atomic force microscopy (AFM),17 and grazing incidence X-ray diffraction (GIXD).6J8J9The first two techniques provide good information on local monolayer morphology (however, with resolution not better than 1pm) but no data about a crystalline structure. AFM improves remarkably the spatial resolution but only when the monolayer is transferred onto a solid support. Until now GIXD was the only method which allowed the Abstract published in Advance ACSAbstracts, June 15,1994. (1)Weissbuch,I.;Addadi, L.; Leiserowitz,L.; Lahav, M. J.Am. Chem. SOC.1988,110, 561. (2) Landau, E. M.; Grayer-Wolf, S.; Sagiv, J.;Deutsch, M.; Kjaer, IC; Als-Nielsen, J.;Leiserowitz, L.; Lahav, M. Pure Appl. Chem. 1989,61, 673. (3) Landau, E. M.; Levanon, M.; Leiserowitz, L.; Lahav, M.; Sagiv, J. Nature 1985, 318, 353. (4) Jacquemain, D.; Leveiller, F.; Weinbach, S. P.; Lahav, M.; Leiserowitz, L.; Kjaer, K.; Als-Nielsen,J. J.Am. Chem. SOC.1991,113, 7684. Leveiller, F.;Jacquemain,D.; Lahav, M.; Leiserowitz,L.; Deutsch, M.; Kjaer, K.; Als-Nielsen, J. Science 1991,252, 1532. ( 5 ) Jacquemain, D.; Grayer-Wolf,S.; Leveiller,F.; Deutsch, M.; ylaer, K; Als-Nielsen, J.; Lahav, M.; Leiserowitz, L. Angew. Chem., Znt. Ed. 1992. 31. 130. E n d . ~-~ (6) Landau, E:M.; Popovitz-Biro, R.; Levanon, M.; Leiserowitz, L.; Lahav, M. Mol. Cryst. Liq. Cryst. 1986, 134, 323. (7) Mann, S.;Hewood, B. R.;Raiam, S.;Birchall,J. D. Nature 1988, 334, 692. (8)Weissbuch, I.; Berkovic, G.; Lahav, M.; Leiserowitz, L. J . Am. Chem. SOC.1990,112, 5874. (9) Gavish, M.; Popovitz-Biro, R.;Lahav, M.; Leiserowitz, L. Science 1990,250,973. (10)Heywood, B. R.; Mann, S. J . Am. Chem. SOC.1992,114,4681. (11)Heywood, B. R.; Mann, S. Langmuir 1992, 8, 1492. (12) Henon, S.; Meunier, J. Rev. Sci. Znstrum. 1991, 62, 936. (13) Honig, D.; Mobius, D. Thin Solid Films 1992,210/211, 64. (14) Weiss. R. M.: McConnel. M. Nature 1984. 310, 47. (15)Losche,M.; Sackmann, E.; Mohwald, H. Ber. Bu'men-Ges. Phys. Chem. 1984,87,848. (16) Bere. B. Nature 1991.322. 350. (17) Schkartz, D. K.; Garn'aes, J.; Viswanathan, R.;Zasadzinski, J. A. N. Science 1992,257, 508. (18)Als-Nielsen, J.; Christensen, F.; Pershan, P. S. Phys. Rev.Lett. @
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1982 - _ _ _ 48 - - , 1107 I
(19) Kjaer, K;Als-Nielsen,J.;Helm, C. A.; Laxhuber, L. A.;Mohwald, H. Phys. Rev.Lett. 1987, 58, 2224.
monolayers to be studied in situ and their crystallographic structure to be obtained with near-atomic r e s ~ l u t i o nIt. ~ yields, however, absolutely no information on the monolayer morphology. Recently we have demonstrated a new approach,2Ocryotransmission electron microscopy (cryo-TEM),for probing local crystallographic structure and morphology of amphiphilic monolayers simultaneously almost in situ. This method is based on fast freezing the monolayer together with a thin layer of water subphase. The water transforms into vitreous ice, and the structure of the monolayer is preserved as being on water. Thus, we were able to investigate successfullyboth the crystalline structure and morphology of several monolayer systems such as longchain alcohols, acids, and acid salts. The obtained results correlated well with GIXD data. Nevertheless, the question remained whether the presence of a plastic support underneath the layer of vitreous ice has an influence on the crystalline structure of the monolayers investigated. A possible way to answer this question was to deposit the monolayer directly on a grid without support. Such an experiment was performed using the smallest commercially available grid of 1000 mesh.21 However, a disadvantage of this approach was the difficulty of obtaining a homogeneous and thin layer of vitreous ice.22 In this report we suggest a way to overcome this difficulty by using plastic holey grids with holes 5-7 pm in size.
Experimental Section Plastic holey grids23were used as a support for monolayer films to be deposited. Grids were treated by glow discharge (to make them more hydr0philic2~)and placed on a stainless steel mesh, in a specially prepared Teflon trough, under the water surface. In all the experiments Millipore-purified water was used. The top surface of water was thoroughly cleaned prior to monolayer deposition. Monolayers were spread using a -5 x M chloroform solution on the water surface (with a coverage of -70%) at 20 "C.The subphase was then cooled to 5 "C, as GlXD studies had shown that the monolayers in the relaxed state (Le., correspondingto -70% coverage)are highly crystalline (20)Majewski, J.;Margulis, L.;Jacquemain, D.; Leveiller, F.; Bohm, C.; k a d , T.; Talmon, Y.; Lahav, M.; Leiserowitz, L. Science 1993,261, 899. (21) Weissbuch, I.; Majewski, J.;Margulis, L.; Lahav, M.; Leiserowitz J. Phys. Chem. 1993,97, 8692. (22)The mesh size (-15 pmJ wa8 too big to provide a thin layer of ice with constant thickness. (23)Robards, A. W., Wilson, A. J., Eds. Procedures in Electron Microscopy; John Wiley & Sons: New York, 1993; pp 4:6, 15. (24) Dubochet, J.; Groom, M.; Mueller-Neudeboom, S. In Aduances in Optical and Electron Microscopy; Barer, R., Cosslett, V. E.,Eds.; Academic Press: New York, 1982; Vol. 8, pp 107-135.
0743-746319412410-2081$04.50/0 1994 American Chemical Society
2082 Langmuir, Vol. 10, No. 7, 1994
Letters
Figure 1. Bright-field image and diffraction patterns from the monolayer of C31H630H deposited on a holey plastic film. The monolayer is supported by a thin layer of vitrified water. (a)shows a micrograph which shows a continuous membrane with holes.
Note that some of the holes have different than round shape, displaying distinct facets. The brighter, circular region marks the selected area from where ED patterns, shown in (b) and (c), were obtained. In (b) three sharp rings represent the diffraction from a n orthogonal packing of hydrocarbon chains in the rectangular unit cell. They were indexed as { l,l},{ 0,2},and { 2,0}, respectively. (c) shows diffraction from the same area after -10 s of prior exposure to the electron beam, which destroyed the crystallinity of the monolayer. One can see that the sharp rings of the monolayer disappeared, leaving only two diffised rings, indicating the presence of vitreous ice.
at that temperature. The water subphase was slowly drained with a motor-driven syringe. The grids with the monolayer depositedwere transferred to the plunging device.% Excess water on the underside of the grid was blotted with filter paper, and the grids were rapidly plunged into liquid ethane cooled with liquid nitrogen to its freezing point, using a "guillotine". Such fast cooling induced vitrification of the thin layer of water subphase and allowed the water-monolayer interface inside the holes of the grid to be preserved in situ. As a rule the thickness of the vitrified water layer varied inside individual meshes, and in many cases it was thin enough to be transparent for electrons.
The electron -action (ED)patterns of vitreous water displayed two diffise rings corresponding to two d spacings of 3.70 and 2.14A as reported before26and did not interfere with dieaction from the monolayers. The grids were loaded into a Gatan 626cold stage and examined in a Philips CM12 transmission electron microscope operated a t 100 kV. The samples were maintained a t -175 "Cthroughout their examination. To minimize radiation damage, low-dose precautions were used. The radiation dose was approximately 5 electronsh", which is close to the values used earlier for studying other surfactant m0nolayers.2~
(25) Bellare, J. R.; Davis, H. T.;Scriven, I. F.;Talmon,Y.J . Electron Microsc. Tech. 1988,10, 87.
(26) Dubochet, J.; Adrian, M.; Chang, J. J.; Homo, J. C.; Lepault, J.; McDowall, A. W.; Schultz, P. Rev. Bwphys. 1988,21, 129.
Letters Observedreflectionsfrom vitreous (and hexagonal, albeit rare) ice were used to calibrate the camera length and so permit the d spacing to be determined with sufficient accuracy.
Results Here we present the results of cryo-TEM experiments with monolayers of the amphiphilic alcohol C31H630H. This monolayer has already been shown by GMDz8to be highly crystalline in the relaxed state on the water surface. Also, TEM observations on vitrified samples with a plastic support confirmedthat the 2-D crystalline domains remain intact upon vitrification.z0 A typical bright-field image of the C31H630H monolayer deposited onto the holey grid and vitrified is shown in Figure la. A bright circle in Figure l a shows the size and position of the selected area from where ED patterns (Figure lb,c) were taken. In Figure l b two diffuse reflections from vitreous ice are visible as broad halos. Three sharp rings correspond to the monolayer and may be indexed as {l,l}, {0,2} and (2,O) reflections with d spacings of 4.2, 3.7, and 2.5 A, respectively. Such assignment yields a rectangular unit cell with dimensions ofa = 5.05Aand b = 7.4Aandanareapermolecule(ab/2) of 18.7 A2. The cell dimensions provide fingerprint evidence that the two hydrocarbon chains in the unit cell are related to each other by glide symmetry such that the plains through their carbon backbones make an angle of -90°.29 This arrangement has been specified as the orthogonal 01motif. The diffraction rings indicate that the crystallites are randomly oriented azimuthally. The inner ring shows more diffraction spots than the outer one, in keeping with the 2: 1multiplicity ratio of the { 1,1} and {0,2} reflections. One can observe that no more than 10-20 crystalline domains participate in diffraction. Therefore, an average domain size may be estimated as 0.5-1.0pm, taking into account the selected area diameter (3pm, see Figure la). The crystalline structure of the monolayer appeared to be extremely sensitive to the electron beam irradiation. (27) Garoff, S.;Deckman, H. W.; Dunsmuir, J. H.; Alvarez, M. S.; Bloch, J. M. J.Phys. (Paris) 1986, 47, 701. (28) Majewski,J.;Popovitz-Biro,R.; Kjaer,IC;Als-Nielsen,J.; Lahav, M.; Leiserowitz, L. J.Phys. Chem. 1994, 98, 4087. (29) Small, D. M. Handbook o f l i p i d Research; Plenum: New York, 1986; Vol. 4.
Langmuir, Vol. 10, No. 7, 1994 2083 It is destroyed in less than 10 s even under low-dose precautions. Such an amorphization results in diffuseness and finally total disappearance of diffraction rings. A typical example of this effect is given in Figure IC.This pattern was obtained exactly from the same selected area as the diffraction pattern shown in Figure l b but after -10 s of additional exposure to the electron beam. One can clearly see that the Bragg reflections from the monolayer totally disappeared while the diffused diffraction rings of vitreous ice remained with no changes. Bright-field imaging of the monolayer on vitreous ice (Figure la) revealed the presence of a continuous film with holes several tenths of a micrometer in size. These holes appeared to be only slightly brighter than the film itself. Some of them had well-defined facets which indirectly reflect faceting of crystalline domains (domain boundaries are invisible in the bright-field images, but can be recognized in the dark-field mode, as was shown previous1y2O). This observation was supported by our atomic force microscopy measurement^;^^ a similar observation was also reported for monolayers of stearic acid.31 In previous studies of C31H630Hmonolayers on supported gridsz0it was difficult to observe the monolayer domains in bright-field mode. In the present case the phase contrast was substantially enhanced by using the large underfocus (100-200 pm) for recording the images. A lack of the support film contributed also to the improvement of an image of the membrane which is only on a thin layer of vitreous ice.
Conclusions The presented technique of sample preparation using plastic holey grids was found to be complementary to those used previously but has additional advantages. Firstly, it allows a thin and homogeneous layer of vitreous ice to be obtained underneath an investigated monolayer. Secondly, it eliminates possible sources of artifacts connected with the presence of a plastic support. Due to these properties the proposed technique seems to be very suitable for studying thin organic films on aqueous subphases by cryo-TEM. (30) Atomic force microscopy revealed a very similar continuous membrane of monolaver with faceted holes 0.5-3 um in size. (31) Ueda, N.;Takenaka, T.; Aoyama, R;Matsumoto, M.; Fujioshi, Y. Nature 1987,327, 319.
