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Cryogenic Electron Microscopy of Micelles and Lyotropic Liquid Crystals in Some Polar Solvents Z. Lin,† H. T. Davis,* and L. E. Scriven* Department of Chemical Engineering & Materials Science, University of Minnesota, 421 Washington Avenue S.E., Minneapolis, Minnesota 55455 Received February 9, 1996. In Final Form: June 24, 1996X Micelles and lyotropic liquid crystals in the polar solvents glycerol and formamide have been imaged at high resolution by cryo-transmission electron microscopy (cryo-TEM) of thin frozen samples. Sample films were prepared in a chamber in which temperature and component chemical activities in the surrounding vapor were controlled. The controlled evaporation of the solvent on the grid, the so-called on-grid processing technique, was used to prepare the viscous liquid crystal phase. In dilute solutions of cetyltrimethylammonium bromide (CTAB) in glycerol and sodium dodecyl sulfate (SDS) in formamide there were spherical micelles, while in the concentrated solution of CTAB in glycerol hexagonal liquid crystals formed. These structures resemble closely in appearance the corresponding structures in aqueous solution. Electron beam radiation damage is much more severe in nonaqueous solutions, and the contrast is much lower.
Introduction Amphiphiles are molecules that possess a dual nature; i.e., polar and nonpolar portions are covalently bonded, and the former have affinity for a polar environment whereas the latter have affinity for a nonpolar environment. When the bonding of these portions allows them to be opposite ends of a wandlike or straplike molecule, the amphiphiles may organize side-by-side into oriented surfacelike structures that reduce contact between nonpolar ends and polar environments, polar ends and nonpolar environments, or both, thereby reducing the total free energy of the system. The two sides of the surfacelike microstructure consist of each of the ends of the amphiphiles. The surfacelike aggregates can take on a variety of forms, such as monolayers, globular or rotund micelles, tubelike or wormlike micelles, bilayers, and lyotropic liquid crystals, including (i) hexagonal phase, ordered assemblages of tubelike micelles, (ii) lamellar phase, assembled bilayers; and (iii) cubic phases, ordered assemblages of globular micelles and in some cases a nonglobular cubic phase in between the hexagonal and lamellar phases. The topology, size, and shape of the aggregates formed, whether in water or another solvent, depend on not only the chemical and physical nature of the surfactant but also concentration, pressure, temperature, pH, ionic strength, and so on. The way these factors balance out is measured by the free energy, to which they all contribute. At equilibrium, the free energy is a minimum. The minimum may belong to a molecular solution, a micellar solution, a liquid crystalline or crystalline phase, or two or more coexisting phases. The free energy can of course be broken into an enthalpic part that derives directly from intermolecular forces and an entropic part that derives from configurations and arrangements accessible by thermal motions. For example, micelle formation in the polar solvent hydrazine is an enthalpy-driven process, whereas in water it is entropy-driven.1 Cryogenic transmission electron microscopy (cryo-TEM) is a method whereby the specimen can be examined in its † Current address: Miami Valley Laboratories, The Procter & Gamble Company, P.O. Box 538707, Cincinnati, OH 45253-8707. * To whom correspondence should be addressed. X Abstract published in Advance ACS Abstracts, October 1, 1996.
(1) Evans, D. F.; Ninham, B. W. Molecular force in the selforganization of amphiphiles. J. Phys. Chem. 1986, 90, 226-234.
S0743-7463(96)00122-9 CCC: $12.00
native state by thermal fixation. Cryo-TEM of the surfactant aggregates that form in aqueous solutions has been achieved by microscopists. It can provide more information about these aggregates at high resolution and with fewer artifacts than some other imaging methods, such as freeze-fracture and staining techniques. Recent developments of sample preparation have allowed surfactant aggregates to be visualized by electron microscopy. It is now well established that thermal fixation of thin liquid films of aqueous systems of biological and synthetic materials produces the fewest artifacts among sample preparation techniques for transmission electron microscopy (TEM). Direct imaging of the thermally fixed specimen provides high-resolution information for systems like viruses,2 vesicles, micelles, and ripple phases.3-5 Because microstructures formed in aqueous systems are temperature and concentration dependent, a controlled environment vitrification system (CEVS) developed by Bellare et al.6 was designed for sample preparation of these systems. The CEVS has been used to study the microstructure of many association colloids at equilibrium.4,5 The further development of the so-called time-resolved cryo-TEM and on-grid processing make it possible to study nonequilibrium states of evolving systems by halting them in midprocess.7 This approach in turn makes it possible to use cryo-TEM to image intermediate structures formed during chemical or physical changes.8 Lyotropic liquid crystal phases in nonaqueous solvents have been studied by the freeze-fracture transmission electron microscopy technique.9,10 However, despite all (2) Adrian, M.; Dubochet, J.; Lepault, J.; McDowall, A. W. Cryoelectron microscopy of viruses. Nature 1984, 308, 32-36. (3) Talmon, Y. Imaging surfactant dispersions by electron microscopy of vitrified specimens. Colloids Surf. 1986, 19, 237-248. (4) Clausen, T. M.; Vinson, P. K.; Minter, J. R.; Davis, H. T.; Talmon, Y.; Miller, W. G. Viscoelastic micellar solutions: Microscopy and rheology. J. Phys. Chem. 1992, 96, 474-484. (5) Vinson, P. K.; Bellare, J. R.; Davis, H. T.; Miller, W. G.; Scriven, L. E. Direct imaging of surfactant micelles, vesicles, discs and ripple phase structures by cryo-transmission electron microscopy. J. Colloid Interface Sci. 1991, 142, 74-91. (6) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon, Y. Controlled environment vitrification system (CEVS): An improved sample preparation technique. J. Electron Microsc. Tech. 1988, 10, 87-111. (7) Talmon, Y.; Burns, J. L.; Chestnut, M. H.; Siegel, D. P. Timeresolved cryotransmission electron microscopy. J. Electron Microsc. Tech. 1990, 14, 6-12. (8) Siegel, D. P.; Burns, J. L.; Chestnut, M. H.; Talmon, Y. Intermediates in membrane fusion and bilayer/nonbilayer phase transitions imaged by time-resolved cryo-transmission electron microscopy. Biophys. J. 1989, 56, 161-169.
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the successes with aqueous systems, cryo-TEM imaging of microstructures in nonaqueous solvents has not been reported. In fact, although there are some reports recently on aggregates of surfactant in nonaqueous polar solvents by other techniques,11-13 micelle formation in nonaqueous polar solvents has not been explored microscopically. More conclusive evidence of micelle formation is needed. The purpose of this work is to image micelles and lyotropic liquid crystals in nonaqueous, polar solutions. These images provide the conclusive evidence of micelle formation in polar solvents. Contrast and electron beam radiation damage are major difficulties to get these images. This work also provided the first-hand data of radiation damage in polar solvents. Materials and Methods Cetyltrimethylammonium bromide (CTAB) (99%) was purchased from Eastman Kodak Co., Rochester, NY. Sodium dodecyl sulfate (SDS) (>99%) was obtained from BDH, U.K. Glycerol (>99%) and formamide (>99%) were obtained from Aldrich, Milwaukee, WI. All the chemicals were used as received. All the solutions were prepared by adding solvent into weighed surfactant to the desired weight percent. All the samples were heated to 60-90 °C so that the surfactant was totally dissolved into the solvent. Samples for cryo-TEM were prepared in a CEVS, which has been described in detail elsewhere.6 In short, the CEVS is an environmental chamber in which temperature can be controlled within 0.1 °C between -10 and +90 °C by a 600 W halogenquartz lamp and an insulated reservoir mounted on the outside rear wall of the chamber. The reservoir can be charged with a refrigerant. The atmosphere inside can be saturated by the solvent(s). This is accomplished with porous sponges extending upward from liquid reservoirs. The air inside the chamber is recirculated across the sponges by a fan mounted on the back wall of the chamber to reduce the temperature and composition gradients in the vapor. In the studies reported here, thin films of sample were formed by placing a 5 µL drop of the surfactant solution on a holey polymer support film which had been coated with carbon and mounted on the surface of a 200-mesh standard TEM grid.14 The grid was held by a tweezer mounted on the plunge assembly. The drop was then blotted with filter paper from both sides of the grid to remove the excess liquid by moving the filter paper up and down while it was gently touching the grid surface. After most of the liquid had been removed, the rest formed a thin film spanning the holes in the holey polmer film. The holes had typical sizes of 1-10 µm, and the suitable film thickness was less than 200 nm. The suitable film thickness could be gotten by trial-anderror. One way to make sure that the film formed on the grid had a suitable thickness was to leave some liquid on the mesh which was close to the tweezer so that it could be seen by eye while blotting the other side of the grid harder so that there was a thickness gradient on the grid. This ensured that there would be a film with a suitable thickness on the grid. The entire assembly was then plunged through a synchronous shutter at (9) Abiyaala, M.; Duval, P. Freeze-fracture electron microscopy study of hexagonal phase defects in a sodium dodecyl sulfate-formamide system. J. Phys. II 1994, 4, 1687-1698. (10) Auvray, X.; Abiyaala, M.; Duval, P.; Petipas, C.; Rico, I.; Lattes, A. X-ray diffraction and freeze-fracture electron microscopy study of the cubic phase in the cetylpyridinium chloride/formamide and cetyltrimethylammonium chloride/formamide systems. Langmuir 1993, 9, 444-448. (11) Auvray, X.; Perche, T.; Anthore, R.; Petipas, C.; Rico, I.; Lattes, A. Structure of lyotropic phases formed by sodium dodecyl sulfate in polar solvents. Langmuir 1991, 7, 2385-2393. (12) Jonstroemer, M.; Sjoeberg, M.; Warnheim, T. Aggregation and solvent interaction in nonionic surfactant systems with formamide. J. Phys. Chem. 1990, 94, 7549-7555. (13) Warnheim, T.; Joensson, A. Phase diagrams of alkyltrimethylammonium surfactants in some polar solvents. J. Colloid Interface Sci. 1988, 125, 627-633. (14) Vinson, P. K. The preparation and study of a holey polymer film. Proceedings of the 45th Annual Meeting of the Electron Microscopy Society of America; Bailey, G. W., Ed.; San Francisco Press: San Francisco, 1987; pp 644-645.
Lin et al. the bottom of the environmental chamber and into the liquid ethane situated immediately beneath it. The plunge velocity is high (>2 m/s) and reproducible. Ethane was chosen as the cryogen because the large temperature difference between its melting and boiling points helps prevent the formation of a vapor envelope around the specimen which would slow down the freezing process during the fixation. The geometry of the specimen, which had a large surface area and a small thickness, the thermodynamic properties of ethane, and the high plunge velocity provide a cooling rate which is high enough to prevent ice crystals from forming. This prevents possible structural rearrangements associated with thermotropic phase transitions within the system. The frozen specimen was transferred under liquid nitrogen into a liquid nitrogen-filled Dewar for storage. One of the problems associated with blotting is it requires the solution to be a shear-thinning liquid and to have a low viscosity. In the case of liquid crystalline materials, which usually have a higher viscosity than the micellar solution, the blotting becomes somewhat difficult. It is not easy to thin the film down to the suitable thickness without pressing the grid very hard, which could destroy the holey ploymer film on the grid. However, one can circumvent this difficulty by adopting the concept of on-grid processing;7 a high-viscosity liquid crystal sample can be prepared from a low-viscosity dilute solution, usually a micellar solution, by on-grid evaporation. This method was used to form gels on grids from organic polymer solution,15 and from polymerizing solutions of inorganic polymer precursors called sols.16 Although the surfactant concentration in the specimen that is examined is not known exactly, it can be estimated well from the known composition of the liquid crystalline phase in equilibrium with the micellar phase. This is the way we prepared the liquid crystal specimen. A low-viscosity micellar solution of high enough surfactant concentration that it is close to the liquid crystal phase was placed on a holey carbon grid and was blotted as previously described. Then the solvent was evaporated from the sample for about 30 min at 90 °C in the environmental chamber with the help of the fan in the chamber. This increased the surfactant concentration in the sample and achieved a liquid crystal phase. The specimen then was frozen by the method mentioned before. The frozen specimen was loaded under liquid nitrogen into the cryo-TEM transfer stage (Model 626, Gatan, Inc., Warendale, PA), and the holder was inserted into the microscope (JEOL 1200EXII or JEOL 1010, JEOL U.S.A., Boston, MA) for observation. The microscope was operated at 120 kV (JEOL 1200EXII) or 100 kV (JEOL 1010). The cryoholder temperature was maintained below -170 °C during imaging. The condensing lens aperture was set at 100 µm in diameter, and the objective aperture was 50 µm in diameter. The specimen was imaged at a nominal underfocus of 2-4 µm in order to achieve adequate phase contrast. The images were recorded on the Kodak SO-163 film at 20 00030 000× ((5%), 1 s exposure time, and 2-6 pA/cm2 screen current. The film was developed with full-strength D-19 developer (Eastman Kodak, Co., Rochester, NY) for 12 min.
Results and Discussion Figure 1 shows rotund, ‘spherical’ micelles of CTAB in both glycerol (Figure 1a) and water (Figure 1b). The micelles appear to be about the same size in both media. However, the contrast of the micelles in glycerol is lower than that of those in water. (15) Cohen, Y.; Talmon, Y.; Thomas, E. L. On the structure of poly(γ-benzyl-L-glutamate) (PBGL) gels. In Physical Networks; Burchard, W., Ross-Murphy, S. B., Eds.; Elsevier Applied Science Publishers: Amsterdam, 1990; pp 147-158. (16) Bellare, J. R.; Bailey, J. K.; Mecartney, M. Freezing dynamical sol-gel processes with the controlled environment vitrification system. Proceedings of the 45th Annual Meeting of the Electron Microscopy Society of America; Bailey, G. W., Ed.; San Francisco Press: San Francisco, 1987; pp 356-357. (17) Sjoeberg, M.; Henriksson, U.; Warnheim, T. 2H nuclear magnetic relaxation of [1,1-2H]hexadecyltrimethylammonium bromide in micellar solutions of nonaqueous polar solvents and their mixtures with water. Langmuir 1990, 6, 1205-1211. (18) Rico, I.; Lattes, A. Formamide, a water substitute. 12. Krafft temperature and micelle formation of ionic surfactants in formamide J. Phys. Chem. 1986, 90, 5870-5872.
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Figure 1. (a) Rotund, globular micelles of 1.8% CTAB in glycerol at 60 °C. (b) Rotund, globular micelles of 4% CTAB in water at 25 °C.
Figure 2. (a) Rotund, globular micelles of 28.3% SDS in formamide at 60 °C. (b) Rotund, globular micelles of 10% SDS in water at 25 °C.
Figure 3. (a) Micellar liquid crystal, presumably hexagonal phase of CTAB in glycerol at 60 °C. Tubelike micelles are closely packed; the diameter is the same as that of the spherical micelles. (b)Threadlike micelles of CTAB/4-methylsalicylic acid in water at 25 °C.
It has been reported that the critical micellar concentration (cmc) and the Kraft point of some surfactants were higher in nonaqueous solvents than in water.17,18 This has been taken into account. The CTAB micelles in glycerol were imaged at 60 °C compared to at 25 °C for CTAB micelles in water. Figure 2 shows spherical micelles of SDS in both formamide (Figure 2a) and water (Figure 2b). Again, no differences are apparent, except in the contrast. These images confirm micelle formation in polar solvents. There
is no evidence of an appreciable size difference between micelles formed in formamide and in water. Figure 3a shows an example of an image of CTAB in glycerol prepared by on-grid processing. This is an image of a liquid crystal consisting of arrayed parallel tubes. According to the phase diagram from Warnheim et al.,13 the liquid crystalline phase next to the micellar phase is a hexagonal phase, which is at 50-75% surfactant at 65 °C. The best estimate from the evaporation during the sample preparation indicates the image could very well
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be the hexagonal phase. This hexagonal phase can be viewed as close packed cylindrical micelles. The spacing between the cylinders is about 4.3 nm. Figure 3b shows the tubelike micelles formed by CTAB in aqueous solution. These tubelike micelles are not in the liquid crystal state; they aligned themselves along the flow directions during the sample preparation for the cryo-TEM study. The apparent diameter of the micelles in glycerol is smaller than that in water. However, the diffferent defocus condition can distort the results. One of the difficulties of imaging surfactant aggregates in solution is electron beam radiation damage. Radiation damage refers to structural or compositional changes of the specimen by electron beam irradiation. Radiation damage is inevitable in the electron microscopy because it is part of the electron beam-specimen interaction that is responsible for forming the image. The electron dose exposure must be large enough to produce an adequate signal-to-noise ratio and contrast yet small enough to avoid appreciable electron beam radiation damage. Figure 4 shows three images of CTAB in glycerol. The screen currents for Figure 4a and b were 5 and 6 pA/cm2, respectively. All were taken at 20 000×. The corresponding electron doses on the specimen for 1 s exposure are 125, 150, and 164 e-/nm2, respectively. By taking into account the collection efficiency of about 0.63 for 100 kV,19 the electron doses should be 200 and 240 e-/nm2. According to Echlin,20 for frozen hydrated specimens at 100 K, the electron dose for low-dose imaging is typically 200 e-/nm2. What was found is that, even at such low electron exposure, the smearing effect caused by sample movement (Figure 4a) and the initial stage of solvent (gylcerol in this case) bubbling21,22 (Figure 4b) can be seen. Figure 4c was taken at 6.5 pA/cm2 screen current and 20 000×, which correspond to an electron dose of 260 e-/nm2. The bubbles in Figure 4c are bigger than those in Figure 4b because the higher electron beam exposure. For the successful low-dose imaging example for polar solvents, like Figure 1a and Figure 2a, in which the screen currents were about 3 pA/cm2 at 20 000×, i.e., 120 e-/nm2, after taking the collection efficiency into account, the maximum electron dose allowed in polar solvents is even lower than quoted by Echlin.20 In frozen hydrated specimens, sometimes the aggregates can still be imaged while some radiation damage can be noticed from the bubbling of the supporting holey polymer films. For example, Figure 3b was taken with an electron exposure of 400 e-/nm2, but the tubelike micelles were successfully imaged. According to Echlin,20 the typical electron dose necessary for searching at low dose for good region to image is about 50 e-/nm2. In polar solvents, even under this electron exposure, one still risks damaging the sample, because the electron dose on one specific sample area is the total electron dose on this area, including searching and imaging. A lot of work has been done on radiation damage of aqueous samples by the cryo-TEM technique.21-24 However, there has not been a lot of work done about radiation damage in polar solvents. It appears from the experiments that the radiation damage in polar solvents is much worse than in aqueous solution. (19) Falls, A. H. Electron Microscopy and Molecular Theory of Microstructured Fluids. Doctoral dissertation, University of Minnesota, 1982. (20) Echlin, P. Ice crystal damage and radiation effects in relation to microscopy and analysis at low temperatures. J. Microsc. 1991, 161, 159-170. (21) Talmon, Y. Electron beam radiation damage to organic and biological cryo-specimens. In Cryotechniques in Biological Electron Microscopy; Steinbrecht, R. A., Zierold, K., Eds.; Springer-Verlag: Berlin, 1987; pp 64-84.
Figure 4. (a) 1.8% CTAB in glycerol with an electron dose of 200 e-/nm2. (b) Same as in part a but the electron dose was 240 e-/nm2. (c) Same as in part a but the electron dose was 260 e-/nm2. In parts b and c, solvent bubbling is shown as marked.
As shown in Figures 1 and 2, the contrast of spherical micelles formed in polar solvents is much lower than in aqueous solution. Surfactants, such as SDS and CTAB,
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are made of molecules having relatively light atoms (e.g., carbon, hydrogen, oxygen), as are the solvents. The scattering cross-section of the surfactant aggregates does not differ appreciably from the scattering cross-section of the solvents. Thus, the amplitude contrast is small. Toyoshima et al.25 concluded that the amplitude contrast contribution was less than 7% in their system. The amplitude contrast in polar solvents would be even smaller because the scattering cross-section of polar solvents would be closer to the scattering cross-section of the surfactant aggregate than to that of water. Diffraction contrast arises in crystalline systems but is absent in most vitrified specimens. The dominant mechanism in cryo-TEM of frozen surfactant systems is phase contrast, i.e., differing amounts of phase shift in the electron beam traversing different parts of the specimen. In fact, phase contrast is the main reason spherical micelles can be imaged as shown in Figures 1 and 2. For typical spherical micelles 50-60 Å in diameter, the optimal defocus for a modern electron microscope is about 4 µm. In this case, unlike the typical high-resolution electron microscopy for crystalline materials, the transfer function (22) Dubochet, J.; Adrian, M.; Chang, J.-J.; Homo, J.-C.; Lepault, J.; McDowall, A. W.; Schultz, P. Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys. 1988, 21, 129-228. (23) Glaeser, R. M.; Taylor, K. A. Radiation damage relative to transmission electron microscopy of biological specimens at low temperature: a review. J. Microsc. 1978, 112, 127-138. (24) Lamvik, M. K. Radiation damage in dry and frozen hydrated organic material. J. Microsc. 1991, 161, 171-181. (25) Toyoshima, C.; Unwin, N. Contrast transfer for frozen-hydrated specimens: Determination from pairs of defocused images. Ultramicroscopy 1988, 25, 279-292.
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is almost unaffected by spherical aberration, because this term in the transfer function is much smaller than the defocus term for typical values of the aberration coefficient Cs (about 1-1.5 mm). Of course, every micrograph contains both amplitude and phase contrast. The difference in contrast between micelles in aqueous solution and micelles in polar solvents can come from both the contrast mechanism and also the low electron exposure. As shown in Figure 4, in order to avoid the appreciable electron beam damage, the electron exposure for nonaqueous, polar solvents has to be smaller than for aqueous solution. As result, the signal-to-noise ratio and contrast are lower. In conclusion, micelles of CTAB and SDS surfactants in nonaqueous, polar solvents were imaged for the first time by cryo-TEM. The contrast in these images is low partly because of the low signal-to-noise ratio in order to avoid radiation damage. From the experiments, the maximum electron exposure allowed to image micelles without appreciable electron beam radiation damage in nonaqueous, polar solvents is much lower than in aqueous solution. Acknowledgment. This project was supported by the National Science Foundation Center for Interfacial Engineering at the University of Minnesota. The authors thank Professor Y. Talmon for stimulating discussions and Professor Edward H. Egelman for providing the access to his JEOL 1200 EXII microscope facilities. LA960122Y
