Freeze Fracture Direct Imaging: A New Freeze Fracture Method for

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Freeze Fracture Direct Imaging: A New Freeze Fracture Method for Specimen Preparation in Cryo-Transmission Electron Microscopy L. Belkoura,* C. Stubenrauch, and R. Strey† Institut fu¨ r Physikalische Chemie, Universita¨ t zu Ko¨ ln, Luxemburger Str. 116, D-50939 Ko¨ ln, Germany Received August 22, 2003. In Final Form: February 18, 2004 In this paper, we present a new freeze fracture method for specimen preparation for transmission electron microscopy frozen samples. We call it freeze fracture direct imaging (FFDI) because it is a hybrid of conventional freeze fracture electron microscopy (FFEM) and cryo-transmission electron microscopy (cryo-TEM), combining elements of the fracture technique with direct imaging. Like in FFEM, the sandwich method is used to prepare the sample in a protected fashion. However, after the sample is vitrified and fractured, it is not shadowed but directly imaged. The new technique avoids some experimental artifacts produced by the blotting procedure in conventional cryo-TEM. It relies, though, on occasional fractures transparent to the electrons. The advantageous features are demonstrated by a comparison between conventional cryo-TEM and FFDI micrographs of vesicular solutions. The second outstanding advantage over conventional cryo-TEM is the fact that it is now possible for the very first time to directly image oil-rich mixtures films which normally would dissolve in the cryo-medium ethane. Micrographs of pure oil and of oil-rich microemulsions clearly prove the reliability of the FFDI technique as well as its enormous potential.

1. Introduction With regard to the specimen preparation for the transmission electron microscopy of liquid samples, there are two well-established techniques, namely, the freeze fracture technique freeze fracture electron microscopy (FFEM)1,2 and the direct imaging (DI) of vitrified thin liquid films, that is, the conventional cryo-transmission electron microscopy (cryo-TEM).3-6 Although these two methods are complementary because they elucidate different aspects of the microstructure, a combination of them does not necessarily result in a “real” image of the respective microstructure. We will explain the imperfections of each technique and will present a method which not only combines the advantages of FFEM and cryoTEM but also eliminates some of their respective disadvantages. In FFEM, the specimens are rapidly frozen, fractured, shadowed with metal, and replicated with a thin carbon film. The metal replica of the fracture surface, the morphology of which is controlled by the sample’s microstructure, is then viewed in the transmission electron microscope (TEM). However, it was only 15 years ago that this technique yielded confusing results, namely, different morphologies for the same system. Jahn and Strey7 were * Author to whom correspondence should be addressed. Fax: +49-221-4705104. Tel: +49-221-4704457. E-mail: belkoura@ uni-koeln.de. † Address for reprints. (1) Gulik-Krzywicki, T. Curr. Opin. Colloid Interface Sci. 1997, 2, 132. (2) Meyer, H. W.; Richter, W. Micron 2001, 32, 615. (3) Almgren, M.; Edwards, K.; Gustafsson, J. Curr. Opin. Colloid Interface Sci. 1996, 1, 270. (4) Talmon, Y. In Modern characterization methods of surfactant systems; Binks, B. P., Ed.; Marcel Dekker: New York, 1999; p 147. (5) Almgren, M.; Edwards, K.; Karlsson, G. Colloids Surf., A 2000, 174, 3. (6) Danino, D.; Talmon, Y. In Physical Chemistry of Biological Interfaces; Baszkin, A., Norde, W., Eds.; Marcel Dekker: New York, 2000; p 799-821. (7) Jahn, W.; Strey, R. J. Phys. Chem. 1988, 92, 2294.

the first to visualize the bicontinuous microstructure of a nonionic microemulsion. A few months later, Bodet et al.8 published FFEM images of the same system. Although the interpretation of the images with respect to the microstructure was in perfect agreement with the results of Jahn and Strey,7 the images themselves looked completely different (see Figure 5 for R ) 0.40 in Jahn and Strey7 and Figure 7J in Bodet et al.).8 The differences are discussed in detail in Vinson et al.9 and in Burauer et al.10 What is of relevance for the paper at hand is the fact that Jahn and Strey7 observed cohesive fractures, that is, fractures through the bulk microemulsion, whereas Bodet et al.8 observed adhesive fractures at the metal-microemulsion interface. Although the results complement each other, it is usually the bulk microstructure one is interested in. Thus, to visualize this structure, it is of outstanding importance to use a FFEM technique with which adhesive fractures can either be avoided or distinguished from cohesive ones. The distinction between adhesive and cohesive fractures is possible with a modified version of the well-established sandwich method7,11 proposed by Vinson et al.9 On the assumption that the fractures are cohesive, however, we are faced with yet another problem. In case the structure to be examined does not happen to be fractured, even with a cohesive fracture the microstructure cannot be visualized. In contrast to FFEM, the frozen specimens prepared for conventional cryo-TEM are not replicated but immediately transferred to a low-temperature stage within the microscope and imaged directly. One difficulty in conventional cryo-TEM is the fact that the electron beam (8) Bodet, J.-F.; Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Miller, W. G. J. Phys. Chem. 1988, 92, 1898. (9) Vinson, P. K.; Sheehan, J. G.; Miller, W. G.; Scriven, L. E.; Davis, H. T. J. Phys. Chem. 1991, 95, 2546. (10) Burauer, S.; Belkoura, L.; Stubenrauch, C.; Strey, R. Colloids Surf., A, in press. (11) Willison, J. H. M.; Rowe, A. J. In Practical Methods in Electron Microscopy; Glauert, A. M., Ed.; North-Holland Publishing Co.: Amsterdam, 1980; Vol. 8, p 171.

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damages the sample as has been shown by several scientists.4,12-14 The beam damage is especially severe in the presence of water and even more so in vitrified specimens that contain significant amounts of organic compounds. However, all modern electron microscopes are equipped to perform “minimal dose microscopy”, with which it is possible to handle the strength of the electron beam properly. Indeed, it is not the beam damage but the control of the film thickness which is currently the major difficulty in preparing samples for DI. On one hand, the film has to be thin enough to allow DI. On the other hand, the film has to be thick enough not to influence the microstructure (when the film thickness and the size of the microstructure are similar, confinement effects occur). To obtain the required film thickness, three techniques can be used, namely, the blotting of the sample prior to vitrification,6 the preparation of cryo-sections of vitrified samples,15 and the preparation of films for which the thickness is controlled interferometrically before vitrification.16,17 Because the latter two methods are still difficult to handle, they are not used by many researchers; the former, however, is widely applicable and, thus, wellestablished. Nevertheless, it has one major disadvantage, namely, the occurrence of preparation artifacts induced by the blotting procedure. Blotting does not only lead to inevitable concentration changes of the thinned sample drop (the amount of solvent and dispersed phase adsorbed by the paper are not the same) and to size segregation but also to the shearing of internal liquid structures.18 The experimental problems are described by Talmon:4 “The exact amount of blotting and its mode, i.e. how much shear is applied on the liquid, depends on the liquid. The operator has to adjust the blotting based on his or her experience with that particular system.” Obviously, the blotting procedure is not the ideal way to get reproducible images of the microstructure. In addition to the above-mentioned artifacts, the blotting procedure is restricted to solutions of low viscosity. Last but not least, one great disadvantage of conventional preparation for cryo-TEM is the fact that in its present form it cannot be applied to liquids which are soluble in the cryogen. Because the cryogen of choice is liquid ethane (L Eth) or propane, most organic solvents will be dissolved during the preparation and a visualization of the respective oil-continuous structure is impossible.4 In conclusion, we are still faced with the experimental challenge of guaranteeing a real image of the structure to be examined. It has been pointed out above that with the FFEM technique the information about the structure depends on the quality of the fracture, whereas with conventional cryo-TEM the “blotting” leads to inevitable artifacts produced by size segregation, the shearing of the sample, or by concentration changes. Moreover, so far oil-rich samples can only be visualized by FFEM and cannot be imaged directly. To overcome these problems we developed a new method which is called freeze fracture direct imaging (FFDI). FFDI is a hybrid of FFEM and cryo-TEM. Like in the FFEM technique, the sample is freeze-fractured. However, instead of replicating the fracture, it is the fracture itself that is looked at with the microscope (DI). In the paper at hand, we will present (12) Talmon, Y. J. Microsc. 1986, 141, 375. (13) Echlin, P. J. Microsc. 1991, 161, 159. (14) Lamvik, M. K. J. Microsc. 1991, 161, 171. (15) Dubochet, J.; Blanc, N. S. Micron 2001, 32, 91. (16) Denkov, N. D.; Yoshimura, H.; Nagayama, K. Ultramicroscopy 1996, 65, 147. (17) Denkov, N. D.; Yoshimura, H.; Nagayama, K.; Kouyama, T. Phys. Rev. Lett. 1996, 76, 2354. (18) Zheng, Y.; Lin, Z.; Zakin, J. L.; Talmon, Y.; Davis, H. T.; Scriven, L. E. J. Phys. Chem. B 2000, 104, 5263.

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the details of the technique (section 2) and demonstrate the strength of FFDI by comparing the resulting images with those obtained by conventional cryo-TEM (section 3) and FFEM (section 4), respectively. 2. FFDI 2.1. Basic Technique. The main goal in the preparation of liquid specimens for TEM is to achieve the formation of thin liquid films spanning the holes of the supporting holey carbon film, which are then vitrified in a cryogen. One of the major problems is to obtain films which are thin enough to be transparent to the electron beam in the microscope. A way to get such thin liquid films without blotting the liquid drop is to place the grid with the holey carbon film between two covering copper plates and to immerse this sandwich assembly into the liquid, like in the preparation procedure for FFEM published by Jahn and Strey.7 The capillary forces make sure that the liquid penetrates the sandwich. The slit of the sandwich is only defined by the thickness of the embedded grid (∼12 µm). The penetration of the liquid in the sandwich further depends on the wetting properties of the liquid under investigation. The embedding of the grid during the preparation not only ensures the protection of the thin liquid film against evaporation but also overcomes all the problems which can be encountered when using the blotting preparation technique.4 Furthermore, this protection is especially important for the preparation of oilrich samples because the sandwich sufficiently protects the sample from the cryogen (L Eth). For the preparation of temperature-sensitive liquid mixtures such as microemulsions, which undergo phase and structure transitions at definite temperatures, a temperature control within the range of (0.02 K is desirable. For that purpose, a special glass tube has been constructed by Burauer et al.10 In combination with a modified plunging device first published by Jahn and Strey7 the required accuracy in temperature can be achieved. The procedure of preparing the specimen for FFDI is as follows: Two copper plates of 0.1-mm thickness (BU 012056-T, Bal-Tec, Lichtenstein) were first coated with a carbon layer (10-40 nm) by evaporating a carbon thread in a sputter coater (SCD 005, Bal-Tec, Lichtenstein) at 0.1 mbar. Depending on the solution to be investigated, hydrophilic or hydrophobic surfaces are needed for the preparation. Thus, to modify the surfaces, the coated copper plates and a copper grid with a holey carbon film (Quantifoil QS 7/2, Jena, Germany) are then exposed in the sputter coater to a glow discharge in an argon atmosphere for 60 s at 0.1 mbar and 10 mA.19 In Figure 1, schematic drawings of the most important preparation steps are shown. First, the copper plates and the copper grid are assembled to form a sandwich, which is held by tweezers (Figure 1, top). Second, the sandwich is inserted in the new developed tube, which is equipped with a temperature sensor (Pt 1000, see Figure 1a). With a stream of dried air (AS), the sandwich is brought to thermal equilibrium, monitored by the temperature sensor Pt 1000. After thermal equilibrium is reached, the air stream is closed (valve V) and the solution (S) is brought into contact with the sandwich (Figure 1b). The solution penetrates into the sandwich, which is then rapidly transferred into the cryogen (L Eth) with the aid of a plunging device (Figure 1c). Because ethane solidifies at the liquid nitrogen (19) Dubochet, J.; Groom, M.; Mueller-Neuteboom, S. In Advances in optical and electron microscopy; Barrer, R., Cosslett, V. E., Eds.; Academic Press: London, New York, 1982; Vol. 8, pp 107-135.

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Figure 1. Schematic drawing of the procedure according to which the specimens of the FFDI technique are prepared. Top: sandwich building. Bottom: (a) glass tube with the solution S, stirrer St, air stream AS, and valve V in a water bath Th; (b) sandwich in contact with the solution; and (c) sandwich in L Eth, which is placed in a copper vessel with a heating wire (HW) and cooled in a Dewar with liquid nitrogen (L N2).

Figure 2. Typical overview FFDI micrograph of a grid where both completely and partly filled meshes with residuals of the fractured bulk phase are clearly seen. The instrument magnification was 250×, and the electron dose was 0.03 e Å-2 s-1. The gray values are modified with a γ1/2 function.

temperature (-196 °C), an electrical heating device (HW) is activated to keep it at its liquid state. Shortly before the transfer, the heating is switched off and the sandwich is plunged into the cryogen just before L Eth starts to solidify. For specimen preparations at room temperature, a cryobox (GA G34-215, Zeiss, Oberkochen, Germany) equipped with a spring-propelled plunging rod was used. After the transfer into liquid nitrogen, the sample is fractured under liquid nitrogen and the grid is mounted in a cryo-holder and viewed in the TEM. Figure 2 shows a typical overview image taken at an instrument magnification of 250×. In the middle of the image, a completely filled mesh (7 µm × 7 µm) can be seen. This mesh is surrounded by only partly filled meshes, in which residuals of the fractured bulk phase are seen. Note that the electron micrograph shown in Figure 4b was taken at the same grid position. 2.2. Extended Technique. Because the new preparation technique is basically a modified method of a wellestablished one used for the preparation of specimens for FFEM,9 it suggested itself to extend this technique to the simultaneous preparation of specimens for FFEM and FFDI, respectively. In this case two grids, namely, one bare grid and one holey carbon film grid, are embedded between two covering copper plates (see Figure 3). The sandwich is dipped into the solution, rapidly frozen, and fractured as schematically drawn in Figure 3. After

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Figure 3. Schematic drawing of the one-step preparation technique for FFDI and FFEM, describing the main steps of the technique: sandwich building, dipping into the solution to be investigated, freezing in L Eth, and fracturing under liquid nitrogen. The sandwich is then separated to directly image one half (FFDI) and to replicate the other (FFEM).

the fracture under liquid nitrogen one grid is used for DI (FFDI) and the second grid, its complementary counterpart, is shadowed with Ta/W and coated with a carbon layer (FFEM).10 The copper plates undergo the same coating and glow discharge procedure as mentioned above before they are assembled to a sandwich. The bare grid used is a hexagonal-mesh tabbed copper TEM grid (Ted Pella, Inc., 8HGC360) 3 mm in diameter. To fix the bare grid on the copper plate, its tab was bent on the copper plate and glued on the back side with a small amount of glue (Pattex WA97, Henkel, Germany) as described in Vinson et al.9 2.3. Cryo-TEM. For the transfer of the sample into the TEM, the sample is mounted on a cryo-specimen holder (CT3500, Oxford Instruments, Oxford, U.K.) precooled at -175 °C, the tip of which is placed in the gas phase of the cryo-box (GA G34-215, Zeiss, Oberkochen, Germany). Once the cryo-specimen holder is inserted into the microscope, the temperature of the cryo-holder controller is set to -145 °C for about 30 min to vaporize off any volatile contaminations, before it is lowered to -168 °C. Inside the microscope, the tip of the cryo-holder which holds the specimen is surrounded by an anti-contaminator metal block cooled with liquid nitrogen reaching a temperature of -175 °C. The TEM (LEO EM 912 Omega) is operated at 120 KV and “zero loss” conditions to enhance image contrast, which is normally affected by inelastically scattered electrons. The acquisition of the electron micrographs is carried out with a cooled 1024 × 1024 charge-coupled device camera (Proscan, Germering, Germany) operating under flat field correction conditions. The software control of the camera was performed with a SIS-image analysis system, version 2.1 (SIS, Mu¨nster, Germany). To avoid beam damage, the search for the vesicles and liquid structures is performed at low magnification (250×) and at a low dose rate of about 0.03 e Å-2 s-1. Higher magnification pictures (10 000×) are taken with a total dose rate ranging between 12 e Å-2 s-1 and 50 e Å-2 s-1 depending on the stability of the specimen position in the electron beam. The respective dose rates are given in the figure captions of each micrograph. Digital image postprocessing has been applied to the electron micrographs if necessary and is mentioned in the respective figure captions of the micrographs. 3. FFDI versus Cryo-TEM As already mentioned in the Introduction, the blotting procedure in the conventional cryo-TEM technique is the source of many artifacts as has been shown by Talmon.4 First, concentration changes are expected if the sample consists of several components. This is due to the adsorption of each component to differing extents onto the filter

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Figure 4. Micrographs (instrument magnification 10 000×) of a physiologically buffered liposome solution containing 1.67 wt % vesicles according to Thomas.20 (a) Conventional cryoTEM technique with blotting at room temperature. The total electron dose was 17 e Å-2. Note the wide variety of vesicle shapes (shear effect) and sizes (size segregation). Moreover, the number density of vesicles is much too high to correspond to 1.67 wt % (concentration change). The gray values are modified with a γ1/2 function. (b) Newly developed FFDI technique without blotting at room temperature. The total electron dose is 50 e Å-2. All vesicles are spherical with a measured mean diameter of 61 ( 11 nm. The gray values are modified with a γ1/2 function.

paper with which the sample is blotted. Second, size segregation occurs. The blotting leads to a flow of the liquid, which, in turn, causes larger objects to move selectively to thicker areas, whereas smaller objects are preferentially left in thinner domains. The thinnest areas may be completely devoid of suspended objects. Third, very high shear rates are applied on the liquid sample while it thins to its final thickness. Such high shear rates may cause microstructural changes in complex fluids. Shearing the sample may not only induce phase transitions but also disrupt, align, or deform aggregates. Faced with such a large number of possible artifacts, one clearly sees that conventional cryo-TEM in its present form is no appropriate technique to quantitatively visualize supramolecular structures. However, with the newly developed FFDI technique described in section 2, all three sources for artifacts have been eliminated because the sample is not blotted. To demonstrate the strength of the FFDI method, images of a liposome solution were made with the conventional cryo-TEM technique (Figure 4a) and with the new FFDI technique (Figure 4b), respectively. The

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buffered solution containing 2 wt % vesicles was sent to us by Thomas.20 Details of the specimen preparation for conventional cryo-TEM are explained in appendix I. The image obtained with the help of the conventional cryo-TEM technique (Figure 4a) shows a highly concentrated vesicle solution. The vesicles differ in size and shape. Elongated, spherical, and peanutlike vesicles of different sizes can be seen. Apart from a great many unilamellar vesicles, a small number of multilamellar vesicles are formed as well. Moreover, it is clearly seen that on average the size of the vesicles seen in the thicker film region (i.e., the darker film region in the bottom left-hand corner of the micrograph) is bigger than in the thinner film region. At first sight, all these observations are surprising. Why is the number density of vesicles far above that expected for 2 wt %? Why is there such a pronounced polydispersity of the vesicles? What is the reason for the size segregation in a liquid which, prior to vitrification, had a homogeneous distribution of sizes and shapes? All these observations can be explained easily by the artifacts produced during the blotting procedure. The contact of the sample with the filter paper obviously leads to a preferential adsorption of the buffer solution. The “zoo” of structures is due to the high shear forces which are applied on the sample while it thins to its final thickness. Last but not least, the size segregation is caused by the different thicknesses of the film prior to vitrification. Claiming that the new FFDI technique solves all the problems mentioned above, one expects an image of a dilute vesicle solution, in which vesicles of constant size and shape are distributed homogeneously. Indeed, such images are easily obtained with the help of the newly developed FFDI technique. Figure 4b shows a reasonable number density of vesicles consistent with the 2 wt %. Size segregation is not seen, and last but not least, the vesicles are spherical and homogeneously distributed. In conclusion, the micrograph of Figure 4a shows a wide variety of vesicle shapes and sizes (shear effect) as well as a too high density of vesicles (concentration change). In Figure 4b, on the other hand, spherical, monodisperse vesicles of an expected number density are seen. The fact that the diameter of these vesicles (61 ( 11 nm) is in agreement with the diameter determined by dynamic light scattering (65 nm)20 proves the reliability of FFDI. The comparison between parts a and b of Figure 4 does not only demonstrates clearly the superiority of FFDI to conventional cryo-TEM but also proves that visualization of the real structures of complex fluids may be difficult by conventional cryo-TEM. 4. FFDI versus FFEM The main drawback of FFEM is the fact that the information about the structure depends on the quality of the fracture. In other words, it is the fracture itself which one looks at to find out about structural details. In contrast, the aim of fracturing the sample in the FFDI technique is simply to obtain a thin, vitrified film that allows DI. Information about the structure is obtained in the next step, namely, by the DI of the fractured sample. It is important to realize that it is not the replicated fracture that is looked at with the TEM but the fractured sample itself. Supposing that the quality of the fracture is satisfactory, FFEM and FFDI appear to be two techniques which complement each other perfectly; while FFEM visualizes the topography of the structure, the internal structure is imaged by FFDI. However, to confirm (20) Thomas, J. E. Ph.D. Thesis, UCLA, Los Angeles, CA, 2001.

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Figure 5. FFDI micrograph (instrument magnification 10 000×) of frozen pure n-octane and the corresponding diffraction pattern (inset in the left upper corner) prepared at room temperature. The arrows in the micrograph point to different thicknesses of the oil film (A < B < C). The inset in the right upper corner shows an overview image (250×) with an arrow indicating the position at which the micrograph (10 000×) was taken. The total electron dose is 1 e Å-2. The gray values are modified with a γ1/2 function.

this assumption, we have to prove that oil-rich samples can be visualized with FFDI too. DI of Pure n-Octane. To demonstrate that oil can be directly imaged with a TEM, an image of pure n-octane was made. It is well-known from the FFEM technique that the cryo-fixation of pure oil in L Eth is possible because the sandwich sufficiently protects the sample from the cryogen (see section 2). Once the sample is frozen and fractured, the task in the FFDI technique is to find a position on the grid where the film is thin enough to allow DI. Figure 5 presents an image of n-octane made with FFDI. The inset in the right upper corner of Figure 5 shows the position on the grid where the image was taken. In Figure 5, it can be seen clearly that n-octane solidifies building layered films of different thicknesses indicated by the white arrows in Figure 5, a point we will get back to later (Figure 8). Furthermore, the oil is structured and shows isolated and connected domains with sizes varying between 27 and 50 nm. The diffuse rings obtained in the electron diffraction pattern (inset in the left upper corner of Figure 5) seem to indicate that the frozen-thin n-octane films have a vitreous or amorphous structure. Assuming alternatively a polycrystalline structure is rather improbable because in that case sharper rings would be expected. Note that with FFEM a decoration of the oil phase is seen when the fracture surfaces are replicated by shadowing with Ta/W.7,10,21 It is this decoration which permits a distinction between oil- and water-rich domains. A look at Figure 5 suggests that the decoration effect simply mirrors the structure of the frozen oil itself. DI of a Water-in-Oil (w/o) Microemulsion. Having proved that it is in fact possible to directly image pure oil, we now should be able to take direct images of any sample, that is, irrespective of the sample composition, and compare them with the results obtained by FFEM. As has been shown in section 2, with the new method a simultaneous preparation of the specimen for both FFEM and FFDI is possible (see Figure 3). After the fracture under liquid nitrogen, one grid is used for DI (FFDI), whereas the second grid, its complementary counterpart, is shadowed and replicated (FFEM). To compare the results of the two techniques, images of a w/o microemulsion (21) Strey, R. Colloid Polym. Sci. 1994, 272, 1005.

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consisting of H2O-n-octane-C12E5 were taken. The composition was chosen to be γb ) 0.05 and wA ) 0.100 (γb ) mC/(mB + mC); wA ) mA/(mA + mB + mC); A ) water, B ) oil, C ) surfactant). At this particular composition and a temperature of T ) 36.34 °C, the microemulsion consists of water droplets in a continuous oil phase. Because of the narrow phase boundaries, an accurate control of the preparation temperature is necessary prior to the cryo-fixation. For the system at hand, a temperature accuracy of (0.02 K is required, which was achieved with the newly developed temperature control unit shown in Figure 1 and described in Burauer et al.10 In Figure 6, the results of the conventional FFEM technique (Figure 6a) and the new FFDI technique (Figure 6b), respectively, are compared. The image obtained with the help of the conventional FFEM technique (Figure 6a, left) shows water droplets in a continuous oil phase. As already mentioned, it is the decoration of the oil that allows us to distinguish between the water- and the oil-rich phases. The diameter of the water droplets is 47 ( 8 nm (see the histogram on the right of Figure 6a) in agreement with the results of smallangle neutron scattering (SANS) measurements carried out by Strey.21 Although this particular sample has not yet been investigated with SANS, Figure 11 in Strey21 allows us to estimate a droplet diameter of 44 ( 5 nm at the preparation temperature of T ) 36.34 °C. The image obtained with FFDI (Figure 6b, left) shows the complementary counterpart to Figure 6a. Note that Figure 6b is the first direct image of a w/o droplet microemulsion, which is not obtainable with conventional cryo-TEM. Comparing part a and part b of Figure 6, one clearly sees that the overall structure is not only qualitatively but also quantitatively the same. From the respective histogram (Figure 6b, right) a diameter of 44 ( 13 nm is obtained for the water droplets, which is in perfect agreement with the FFEM and the SANS results. Slight differences in the size and the number density of water droplets are simply due to the fact that the two micrographs are not taken from the same x,y positions on each grid, that is, that the micrographs do not represent “image” and “mirror image”. In summary, DI of a w/o droplet microemulsion is possible because three major problems have been solved. First, with the FFDI technique all artifacts induced by the blotting procedure can be avoided. Second, the sandwich sufficiently protects the oil from the cryogen. Last but not least, a newly developed setup enables us to reach a temperature accuracy of (0.02 K. With these preconditions, it should be possible to directly image bicontinuous microemulsions as well, which has been impossible so far. DI of a Bicontinuous Microemulsion. As explained above, it is impossible to visualize the structure of a bicontinuous microemulsion with conventional cryo-TEM. However, intensive FFEM studies, the most prominent of which are published in Jahn and Strey,7 Strey,21 and Burauer et al.,10 have shed some light on this structure. With the help of the new method, we aimed to extend the information obtained by the previous FFEM studies by taking direct images of the structure. For that purpose, we again chose the ternary system H2O-n-octane-C12E5, the droplet microemulsion of which has already been successfully visualized above (see Figure 6b). To get a bicontinuous microemulsion, we prepared a mixture consisting of γ ) 0.060 and φ ) 0.50 at T ) 32.62 °C (γ ) mC/(mA + mB + mC); φ ) VB/(VA + VB); A ) water, B ) oil, C ) surfactant). Note that this particular bicontinuous microemulsion has been studied extensively and the respective FFEM images have been published, for example, in Burauer et al.10 In Figure 7, the results of the

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Figure 6. Micrographs (instrument magnification 10 000×) and histograms of a w/o droplet microemulsion of the ternary system H2O-n-octane-C12E5. Sample composition, γb ) 0.05, wA ) 0.100; preparation temperature, T ) 36.34 °C. (a) FFEM, mean diameter of the water droplets 〈d〉 ) 47 ( 8 nm, polydispersity p ) 0.03; (b) FFDI, mean diameter of the water droplets 〈d〉 ) 44 ( 13 nm, polydispersity p ) 0.09. The FFDI image is taken with a total electron dose of 0.51 e Å-2, and the gray values are modified with a γ1/2 function.

conventional FFEM technique (Figure 7a, taken from Burauer et al.)10 and the new FFDI technique (Figure 7b), respectively, are compared. As was the case for the droplet microemulsion, the pictures were taken from the same sample, of which the specimens for both FFEM and FFDI were made simultaneously (see section 2, Figure 3). There is a striking similarity between the structures seen in parts a and b of Figure 7. In Figure 7a, oil-rich and water-rich domains can be distinguished easily because the oil fracture face is specifically decorated. Moreover, it can be clearly seen that the volume fractions of water and oil are equal and that water and oil are arranged in two coexisting phases of equal structure (see Burauer et al.10 for details). A similar situation is seen in Figure 7b. Note that the colors in Figure 7b are inverted to get pictures in which details can be recognized more easily. The digital image processing is explained in appendix II. In the middle of the image, a spongelike structure is formed consisting of a “white” and a “black” subphase. It is important to keep in mind that Figure 7b is a direct image through a sample for which the thickness changes from place to place. Thus, at every position different numbers of layers which all have the structure seen in Figure 7a lie on top of each other. In the middle of the image, where the electron beam passes through the film, the film consists of approximately two to three layers. Because the domain size of this particular microemulsion is approximately 50

nm,22 two to three layers correspond to a film thickness of about 100-150 nm. Toward the upper left-hand and the bottom right-hand corners, the microemulsion seems to be less structured. This observation is due to the fact that the number of layers (i.e., the film thickness) increases, so that details of the structure are no longer visible. A comparison of Figure 7b and Figure 5, however, reveals one inconsistency. Neither of the phases seen in Figure 7b is microstructured, so we have to ask the question of where the oil has gone. The answer to this question is given in Figure 8. Figure 8 is an inverted and color-adjusted image of the bicontinuous microemulsion shown in Figure 7b. The image was taken from another position of the same grid. With the help of the colors, water-rich (pink) and oil-rich (brown) phases can be distinguished. The upper third and the lower part of the image are pure oil films of different thicknesses, whereas in the middle there is a spongelike structure similar to the one seen in Figure 7b. Obviously, part of the oil is expelled from the microemulsion during the cryo-fixation of the sample. This is due to the fact that water solidifies much more quickly than n-octane (at 0 and -57 °C, respectively), so that a rigid water matrix is formed while the oil is still in a liquid state. Thus, as soon (22) Sottmann, T.; Strey, R.; Chen, H.-S. J. Chem. Phys. 1997, 106, 6483.

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Figure 8. FFDI micrograph (instrument magnification 10 000×) of a bicontinuous microemulsion of the ternary system H2O-n-octane-C12E5. Sample composition, φ ) 0.5, γ ) 0.06; preparation temperature, T ) 32.62 °C. The arrows point to regions of expelled oil. The total electron dose is 0.36 e Å-2. Digital image processing is performed according to the description given in appendix II.

Figure 7. Micrographs (instrument magnification 10 000×) of a bicontinuous microemulsion of the ternary system H2On-octane-C12E5. Sample composition: φ ) 0.5, γ ) 0.06; preparation temperature T ) 32.36 °C. (a) FFEM micrograph and (b) FFDI micrograph. The FFDI image is taken with a total electron dose of 0.36 e Å-2. The gray values are modified with a γ1/2 function and inverted.

as the rigid water matrix is formed, the overall structure is not affected by the expulsion of the oil. In conclusion, we have shown that it is indeed possible to directly visualize the bicontinuous structure of a microemulsion. Figures 7b and 8 are the first direct images of the spongelike structure, and their reliability is demonstrated by their striking similarity with Figure 7a. Thus, FFEM and FFDI prove to be complementary techniques which give us a clear picture of the structure. 5. Conclusion The present paper can be regarded as pioneering work because it provides conclusive evidence of the fact that the structure of complex fluids can be directly imaged, avoiding some artifacts known to occur with the conventional cryo-TEM. The newly developed technique is a hybrid of conventional cryo-TEM and FFEM because it combines the fracture technique of FFEM with DI. Like in FFEM, the sandwich method is used to prepare the sample. However, after the sample is vitrified and fractured, it is not shadowed but directly imaged. The main task is to find positions on the grid where the fractured film is thin enough, so that it can be directly imaged. If there is a weak point of our FFDI technique, it is the necessity to find fractured portions which are

thin enough to be penetrated by the electrons. Nevertheless, with FFDI it is possible not only to avoid some artifacts induced by blotting but also to take images of oil-rich samples, because the sandwich prevents direct contact of the cryogen and the oil-rich phase. In the paper at hand, the first direct images of a w/o droplet and a bicontinuous microemulsion are shown. A comparison with the respective FFEM images demonstrates the reliability and strength of the method as well as some advantages relative to conventional cryo-TEM. We have demonstrated that FFEM and FFDI are two techniques which complement each other perfectly: while FFEM visualizes the topography of the structure, the internal structure is imaged by FFDI. After our first report on this technique at the 224th ACS National Meeting in Boston in 2002,23 another research group24 illustrated that FFDI is not only very powerful but also easy to learn after our descriptions23,25 and easy to handle. This research group has published FFDI images of an oil-rich microemulsion and a lamellar phase.24 We consider their work an excellent example of the usefulness of the new FFDI technique. We believe that FFDI (or further developments of it) will soon establish itself among the TEM techniques. Acknowledgment. We would like to thank Dr. S. Burauer for assistance in the early stages of development of the FFDI technique. The technical support of Mr. Metzner and his staff is gratefully acknowledged. Appendix I Preparation of the specimens. A drop (∼3 µL) of the liposome samples is placed onto a holey carbon film (23) Strey, R.; Belkoura, L.; Burauer, S. Bubble formation in supercooled microemulsions; Presented at the 224th ACS National Meeting, Boston, 2002. (24) Agarwal, V.; Singh, M. G.; McPherson, J. V.; Bose, A. Langmuir 2004, 20, 11. (25) Reference 24 in Agarwal et al.24

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Figure 9. FFDI micrograph (instrument magnification 10 000×) of a bicontinuous microemulsion of the ternary system H2On-octane-C12E5. Sample composition, φ ) 0.5, γ ) 0.06; preparation temperature, T ) 32.36 °C. The total electron dose is 0.36 e Å-2. (a) Original electron micrograph, (b) after γ function operation, (c) inverted gray values, (d) applying a DCE filter, (e) RGB colors adjustment, (f) color curves adjustment (corresponds to Figure 8). Note that the water-rich regions are pink, whereas the oil-rich regions are brown.

(Quantifoil S7/2, Jena, Germany) which is held by tweezers mounted on a spring-propelled plunging rod. After the sample drop is thinned by blotting in a saturated environment at 25 °C, the specimen is plunged into a

copper container filled with L Eth at a temperature near its melting point. The copper vessel is placed in the cooled gas phase (-184 °C, measured at the top of the container) of a cryo-box (Zeiss GA G34-215) half filled with liquid

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nitrogen. The vitrified sample is transferred from the L Eth into the cooled gas phase of the cryo-box, freed from remaining L Eth, and stored under liquid nitrogen. Appendix II Digital Image Processing. The quality of electron micrographs obtained from TEM are often very poor as a result of the DI at low electron doses. Note that low electron doses are necessary to prevent the specimen and, thus, the structures from beam damages. A post-digital processing of the micrographs is sometimes necessary to highlight hidden structure details not apparent at the first examination. A series of images are presented in

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Figure 9 to demonstrate how useful the digital image processing can be in visualizing structural details. The gray scales of the original electron micrograph (Figure 9a) are first brightened using a xγ function (Figure 9b) and then inverted (Figure 9c). Using a digital contrast enhancement (DCE) filter of the SIS-Software, version 3.11 (SIS, Mu¨nster, Germany), the weak differences in contrast are emphasized (Figure 9d). The 16-bit gray values image is changed to a 24-bit one to allow RGB colors adjustment (Figure 9e). Finally, the color curves adjustment is performed to end with the image given in Figure 9f. LA0303411