Langmuir 1995,11, 3721-3724
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Cryo-Electron Microscopy of Block Copolymers in an Organic Solvent G. T. Oostergetel,*>' F. J. Esselink,t and G. Hadziioannout BIOSON Research Institute and Laboratory of Polymer Chemistry, University of Groningen, Nyenborgh 4, NL-9747 AG Groningen, The Netherlands Received November 7, 1994. I n Final Form: July 12, 1995@ Solutions of the diblock copolymer polystyrene/poly-2-vinylpyridine(PSP2VP)in toluene were studied by cryo-transmission electron microscopy following fast freezing of a thin film of the solution in liquid nitrogen. The block copolymer forms spherical micelles which can be visualized using phase contrast microscopy. From the measured diameter of copolymer micelles with different block lengths, it can be concluded that only the poly-2-vinylpyridine core is visible in the electron micrographs, whereas the polystyrene corona is not visible and is largely dissolved into the toluene. The block copolymer micelles are rather stable in the beam and provide enough contrast relative to the amorphous toluene, due to the higher density of the micelles relative t o the toluene, to be visible without any staining. This opens the possibility to directly visualize the phase behavior of polymer systems in a volatile solvent, perform timeresolved microscopy, and thus study the dynamics of the formation of different morphologies.
1. Introduction Electron microscopy has become a n important tool in determining the structure and morphology of synthetic polymers. Detailed structural knowledge is indispensable to establish relations between the structure and properties of materials and to understand the processes leading to materials with the desired properties. To date both scanning electron microscopy (SEM) and transmission electron microscopy (TEM) have been used essentially only to study the structure of material in the solid state. However, intermediate stages in the formation of particular structures or morphologies, from a melt or from solution, have been difficult to study by electron microscopic techniques. The main reason for this is the microscope vacuum putting serious limits on the nature of the specimen. In biology a similar problem exists, because in most biological systems water is a crucial component in maintaining the native structure. Most conventional preparation methods for electron microscopy of biological material rely on either carefully removing the water by freeze drying or critical point drying or replacing water by a nonvolatile substance like a resin or a heavy metal salt. Removal of water can be circumvented by using freeze-fracture replication. This technique involves the production of a vacuum-deposited replica from a fracture surface of a frozen specimen. It is well established in cell biology and membrane biology but also applied in areas of materials science, such as colloid Particularly in freeze-fracturing of synthetic polymers, plastic deformation during the cleavage process can lead to hard to interpret artifactual image^.^,^ In all the above mentioned procedures the introduction of preparation artifacts is hard to avoid and interpretation ofthe electron micrographs has to be done with great caution. However, in the last decade low-temperature preparation and microscopical procedures have been developed
* To whom correspondence should be addressed. Telephone: +31 50 634213. Fax: +3150 634800. E-mail:
[email protected]. BIOSON Research Institute. * Laboratory of Polymer Chemistry. Abstract published i n Advance A C S Abstracts, September 15, 1995. (1)Price, C.;Woods, D. Eur. Polym. J. 1973,9, 827. (2) Candeau, F.; Boutillier, J.;Tripier, F.; Wittmann, J . 4 .Polymer 1979,20,1221. (3) Sleytr, U. B.; Robards, A. W. J . Microsc. 1977,110, 1. @
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that allow direct observation of biological material in a close to natural environment. Following fast freezing, tissue can be sectioned and observed in the frozen hydrated state, whereas particles in suspension like viruses or other macromolecular assemblies can be observed directly in a thin layer of vitreous More recently this technique has been extended to the study of amphiphilic molecules in aqueous solution^.^^^ The work described in this paper was prompted by the question of whether cryotechniques similar to those used in biological electron microscopy could be applied to polymer systems comprising a n organic solvent and to elucidate the phase behavior of synthetic polymer systems in solution. Talmon and co-workers showed that it was possible to visualize the structure of poly(y-benzy1-Lglutamate) gels in benzyl alcohol and benzene.8 The system which is being studied here, and used to illustrate the potential capabilities of cryo-electron microscopy in the field of synthetic polymers, consists of the diblock copolymer polystyrene/poly-2-vinylpyridine(PSP2VP). Block copolymers are of great practical interest because they can be used to abolish the incompatibility ofotherwise immiscible polymers. In the case of two immiscible polymers, a diblock copolymer is used where one block is miscible with one polymer and the other block is miscible with the other polymer. The block copolymer reduces the interfacial tension and enhances the mechanical strength of the interfa~e.~JO In the solid state the block copolymers PSP2VP and mixtures with PS were studied by conventional TEM of thin sections.ll It has been shown that above a certain critical concentration (critical micelle concentration) block copolymers form micelles. l2 Obviously this micelle formation is (4) Dubochet, J.; McDowall, A. W. J. Microsc. 1981,124,RP3-4. ( 5 ) Dubochet, J.;Adrian, M.; Chang, J.-J.;Homo, J.-C.; Lepault, J.;
McDowall, A. W.; Schultz, P.Q. Rev. Biophys. 1988,21,129. (6) Talmon, Y. Colloids Surf 1986,19, 237. (7) Frederik, P. M.; Stuart, M. C. A,; Bomans, P. H. H.; Busing, W. M. J . Microsc. 1989,153, 81. ( 8 ) Cohen, Y.; Talmon, Y.; Thomas, E. L. In Physical Networks; Burchard, W., Ross-Murphy, S. B., Eds.; Elsevier Applied Science Pub.: Amsterdam, 1990; p 147. (9) Gaillard, P.; Ossenbach-Sauter, M.; Reiss, G. Makromol. Chem., Rapid Commun. 1980,I , 771. (10) Fayt, R.; JerBme, R.; Theyssie, Ph. J. Polym. Sei., Polym. Phys. Ed. 1981,19, 1269. (11)Esselink, F. J.;Semenov, A. N.; ten Brinke, G.; Hadziioannou, G.; Oostergetel, G. T. Phys. Reu. B 1993,48, 13451. (12) Leibler, L. Makromol. Chem., Macromol. Symp. 1988,16, 1.
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detrimental to the initial goal of acting as a n interfacial agent and lowering the interfacial tension. Theoretical considerations and experimental results on the PS-PS/ P2VP system indicated that block copolymer micelles were formed already in solution, prior to formation of the solid state.ll In order to fully understand the phase behavior of the system and determine the relevant parameters in the formation of different morphologies, i t is important to include the solvent as a crucial factor in the system under study. I t is shown here that i t is indeed possible to study frozen “solutions” of a synthetic polymer in a volatile organic solvent. This opens the possibility to not just visualize morphology but also study “morphogenesis”, the formation of particular morphology.
2. Experimental Section The diblock copolymers used in this study consist of a deuterated polystyrene (PSI block (M,= 75 000) and a poly-2vinylpyridine (P2VP)block (M,= 102 000 and 32 000, respectively), M,/M, < 1.14. The block copolymer was dissolved in freshly distilled toluene yielding a 2% (w/v) solution. A 400 mesh copper electron microscope grid covered with a thin carbon layer can be used (methodA). We also used so called holey carbon films (method B). To obtain these, plain grids were covered with holey Formvar (poly(viny1formal)) support films and subsequently coated with a thin carbon layer. Finally the Formvar was washed away by refluxing for 3 h in acetone vapor. A small droplet of the polymer solution was filtered using a 0.45 pm filter (Sartorius Minisart SRP 15) and applied to the grid. This was done in a cold room (4 “C) in order to limit evaporation of the toluene. Experiments performed a t 20 “C yielded less good specimens because of the fast evaporation of toluene. Most of the liquid is then removed by blotting with filter paper for 2 or 3 s. After blotting, the specimen is immediatelyplunge-frozen in a beaker filled with liquid nitrogen. Liquid ethane or propane (normally used to fast freeze aqueous suspensions) cannot be used because toluene dissolves in these liquids. The grid is transferred under liquid nitrogen and mounted in a precooled Gatan 626 cryo-TEM specimen holder. Micrographs were recorded on Agfa 23D56 film using a JEOL 1200-EXat 120 kV or a Philips CMBOIFEG electron microscope at 200 kV, at nominal magnifications between 1OOOOx and 20 OOOx, and a specimen temperature of -170 “C was used. Electron diffraction was carried out using the Philips CM20/ FEG at 200 kV and a camera length of 340 mm. Mass spectrometry to measure partial pressures of residual gases was performed using a Balzers QMG 064 connected to the specimen chamber of the JEOL 1200-EX.
3. Results and Discussion Cryo-ElectronMicroscopy of Pure TolueneFilms. Thin films of frozen toluene, which is used as a solvent for the PSP2VP block copolymer, are readily formed on holey carbon films (Figure 1). The free spanning films are thin enough to be sufficiently transparent for the electron beam. Using mass spectrometry i t was shown that specimen temperatures below -110 “C are adequate to prevent evaporation of toluene in the high vacuum of the microscope. We noticed that the behavior of frozen toluene in the electron beam is somewhat different from that of frozen water. For hydrated specimens i t is known that water is slowly evaporated under the influence of the beam, leading to a n etching of the specimen. Toluene however, is cross-linked by the electron beam, presumably by a mechanism similar to the formation of hydrocarbon contamination in the microscope. To avoid crystallization of water in hydrated specimens during freezing, cooling rates of > lo4 K s-* are required. This can be achieved if thin (preferably less than a few hundred nanometers) specimens are plunged into a n effective cryogen like liquid ethane or p r ~ p a n e .Since ~
Figure 1. Cryo-electron micrograph of an amorphous frozen toluene film on a holey carbon grid. Bar = 500 nm.
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Figure 2. averaged electron diffractogram of an amorphous frozen toluene film.
(solid) toluene dissolves in liquid ethane, we used liquid nitrogen to freeze the samples. Electron diffraction (Figure 2) indicates that the frozen toluene films are fully amorphous, despite the relatively slow freezing (on the order of lo2- lo7K s-l) in liquid nitrogen. The positions of the maxima in the diffractogram correspond well with the values given by Anderson et al. for amorphous toluene based on X-ray diffraction. Toluene is relatively easy to vitrify,14 but other organic solvents may require faster freezing and hence more efficient cryogens.
Cryo-Electron Microscopy of PSP2VP Micelles in Toluene. The block copolymer was dissolved in toluene. Since this is a good solvent for the PS block but a poor solvent for P2VP block, micelles will form. The core consists of P2VP blocks, while the corona is formed by the PS blocks. In Figure 3, a micrograph is shown of a dPSP2VP block copolymer (75W102K) dissolved in toluene and prepared on a normal carbon support film (method A). The block copolymers have formed micelles which appear as spheres. Only a t the edge of the small (13) Anderson, M.; Bosio, L.; Brunneaux-Poulle, J.; Fourme, R. J. Chima Pltys. 1977, 27, 107. (14) de Nordwall, H. J.;Staveley, L. A. K. Trans. Faruduy Soc. 1956, 52, 1207.
Block Copolymers in an Organic Solvent
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__ - __ Figure 3. micrograph of a solution of dPS/PZVP ('iFjW102K) in toluene at -170 "C, prepared on a normal carbon substrate. White arrows indicate a stepwise increase in thickness of the toluene layer. Black arrowheads indicate micelles not embedded in toluene. Bar 500 = nm. .
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carbon layer
Figure 4. drawing of the quasi-layered structure of PS/P2VP micelles on a carbon substrate.
droplet of the solution on the grid is the layer of toluene thin enough to be able to see the spherical micelles. The idea of micelle-forming block copolymers has been put forward earlier by Tanget al.I5 based on results from light scattering measurements. This, however, we believe, is the first direct view of block copolymer micelles in an organic solvent ever published. Note that the specimens have not been stained and that the micelles are essentially imaged by phase contrast, exploiting the somewhat higher scattering density of (the interior part of) the micelles relative to the toluene matrix.:'.l6.l7 This justifies the interpretation of the micrograph as a direct representation of the situation in solution, contrary to conventional preparation techniques involving fixing and/or drying. From Figure 3 it is clear that the micelles are slightly ordered and cause a stepwise increase in. the thickness of the toluene layer (indicated by the white arrows) going from the edge toward the center of the droplet. In Figure 4 a schematic drawing of the stepwise increase in thickness is given. Furthermore some micelles near the edge of the droplet are not embedded in toluene (indicated by the black arrowheads) and are somewhat flattened. This is probably due to the fact that after the blotting is performed the thin toluene film partly evaporates, even though the temperature is 4 "C. During this evaporation the moving perimeter of the toluene droplet is dragging the block copolymer micelles along. This also explains the apparently high concentration of micelles in the droplet. All the micelles seem to be rather homogeneous in size. The apparent diameter is approximately 48 nm. This value for the diameter is similar to that determined for (15) Tang, W.T.; Hadziioannou, G.; Cotts, P.M.;Smith, B. A. P01~171. Prepr. (Am. Cliem. SOC.,Dia. Polym. Cliem.) 1986,27, 107. ( 16) Handlin, D.L.;Thomas, E. L. Mcrcromolwules 1983,16,1514. (17) Handlin, D.L.;Thomas, E. L. J . Mrrter. Sci. I&. 1984,3, 137.
Figure 5. micrograph of a solution of d P S P 2 W (75W102K) in toluene a t -170 "C, prepared on a holey carbon film. Black arrowheads indicate areas where agglomeration takes place. Bar = 500 nm.
this block copolymer in a homopolymer (PS)matrix.'* From the observed dimension of the micelles and using the known lengths of the two blocks and the density in the solid state, the number of molecules per micelle can be estimated to be in the order of 300 for the 75W102K block copolymer. From that i t can be concluded that the occupation of the PS block at the surface of the P2W core (the interface between the P2W core and the PS outer shell)is far less than unity. The PS moiety must therefore be largely dissolved in the surrounding toluene and is thus not visible in the electron microscope. In the case of P2VPPS micelles in a homopolymer (PS)matrix," the PS moiety is similarly dissolved in the PS matrix. Moreover, in this case only the P2W core is stained with iodine. In Figure 5 a micrograph is shown from the same system but prepared on a holey carbon film (method B). The holes in the carbon support film spanned by the solution are clearly visible. The toluene layer is of a rather uniform thickness which is estimated to be in the order of 100 nm. This estimate is based on the amount of electron scattering from the toluene layer and on the fact that the micelles are somewhat ordered and packed in a single layer, confined to a rather narrow space. The toluene layer can thus be expected to be somewhat thicker than the diameter of the embedded micelles. The edge of the holes in the carbon film clearly affects the packing, which in certain areas is quasi hexagonal. Occasionally micelles seem to fuse into larger agglomerates (see arrowheads in Figure 5). I t is not obvious whether this reflects the situation in solution or is a n artifact introduced during specimen preparation due to the strong spatial confinement of the micelles or due to partial evaporation of the toluene. Figure 6 shows a micrograph of the same specimen as in Figure 5 but from a different area. Again the holey carbon support is visible. The holes, however, are not spanned by a solid toluene film filled with micelles. Instead there seem to be cracks and empty spaces. We ascribe this phenomenon to a fast evaporation of the thin toluene film just before freezing, leaving the block copolymer in a dry state- Actually, this gives an idea about the transition from suspension to the solid state as i t
3724 Langmuir, Vol. 11, NO. 10, 1995
Figure 6. micrograph of a dried film of dPSP2VP (75W102K) a t -170 "C. Bar = 500 nm.
occurs, for example, during the formation of a thin block copolymer film by spincoating. Figure 7, finally, shows a cryo-electron micrograph of a P S P 2 W block copolymer 75W32K in frozen toluene. The micelles are significantly smaller than those in Figure and estimated to be about 30 nm in diameter* This makes sense considering the core micellar dimensions as a function Of the p2VP length* In this micrograph there are also micelles visible lying on the carbon support film. These latter micelles are somewhat larger than those in the holes. This is presumably due to the fact that these are not embedded in and are flattened in a similar way a s those in Figure 3. 4. Conclusions
In this article i t is shown that cryo-electron microscopy is not only useful to study materials in hydrated specimens but can also successfully be applied tosuspensions in an organic solvent (toluene) provided that the material of interest is stable enough in the beam and is able to provide enough contrast.
Oostergetel et al.
Figure 7. micrograph of a solution of dPWP2VP (75W32K)in toluene a t -170 C. Bar = 500 nm.
To avoid (partial) evaporation of the solvent during preparation (between blotting and freezing), a controlled environment vitrification system could be used. lXThis would also allow a proper control of the temperature. I t is shown directly that PSP2VP block copolymers (M\\#(PS) = 75 000, M,(P2VP) = 102 000 and 32 000) form spherical micelles in toluene solutions. These solutions can be readily vitrified. The block copolymer micelles are rather stable in the beam and provide enough phase contrast relative to the amorphous toluene to be visible without any staining. This opens exciting possibilities to directly visualize the phase behavior of polymer systemsin solution. In addition, fast freezing makes i t possible to perform timeresolved' microscopyG and study the dynamics of these processes: morphogenesis, not just morphology. LAM@76H (18) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon, Y. J. Electron Teclt. 19ss, 87. (19) Siegel, D. P.; Burns, J. L.; Chestnut, M. H.;Talmon,Y.BiopAys. J . 1989, 56, 161.
