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J. Phys. Chem. B 2007, 111, 8388-8393
Soft X-ray Microscopy To Characterize Polyelectrolyte Assemblies† Karen Ko1 hler,*,‡ Christophe De´ jugnat,§ Monique Dubois,‡ Thomas Zemb,§,| Gleb B. Sukhorukov,‡,⊥ Peter Guttmann,# and Helmuth Mo1 hwald‡ Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany, Institut de Chimie Se´ paratiVe de Marcoule, CEA Valrhoˆ , Bagnols-sur-Ce` ze, France, Laboratoire Claude Fre´ jacques, DRECAM/SCM, CEA/Saclay, Gif-sur-YVette, France, Department of Materials, Queen Mary UniVersity of London, E1 4NS, London, United Kingdom, and Institut fu¨r Ro¨ntgenphysik, Georg-August-UniVersita¨t Go¨ttingen c/o BESSY, Berlin, Germany ReceiVed: January 16, 2007; In Final Form: February 24, 2007
Transmission microscopy with soft X-rays (TXM) is applied to image in-situ polyelectrolyte assemblies in aqueous environment. The method is element specific and at this stage exhibits a lateral resolution of 20 nm. With the specific examples of hollow capsules and full spheres made of PAH/PSS polyelectrolyte multilayers, it is shown quantitatively that heat treatment irreversibly reduces the water content in the membrane. These experiments complement those reported recently on the polyion system PDADMAC/PSS, which shows a different glass-transition behavior. Finally, the potential and present limitations of TXM are discussed.
Introduction The complexation of polyelectrolytes is important for many of their traditional applications as well as new ones making use of specific supramolecular arrangements in multilayers1-4 and capsules.5,6 Its understanding requires a multitude of techniques which should be applicable in liquid environment and with molecular resolution. These requirements disfavor electron microscopic and other techniques requiring vacuum and have paved the way for optical, infrared, and magnetic resonance spectroscopy to conclude on the local environment of a macromolecule. However, there is generally a strong desire to complement spectroscopic information by data gained in real space. Hence, it is most welcome to develop an element-specific microscopy with resolution down to the single molecular level. In a first study, we have shown that soft X-ray microscopy can be used to investigate the residual water content after heating the sample.7 Using a series of samples where the glass transition was known, we have shown that subsequent heating above that glass-transition temperature of the complex results in the remaining of a lower limit of water content in the complex forming the capsule wall.8 Herein, our aim is to further investigate this water exclusion concomitant to the capsule shrinkage using another polymer system showing a different glass-transition behavior. The absorption of X-rays is known to be element specific, but to make use of this, one needs a tunable X-ray source. This has been introduced in the last 30 years by means of synchrotrons. These powerful light sources are required for X-ray microscopy,9 which has evolved after the development of suitable lenses, that is, Fresnel zone plates, to a powerful † Part of the special issue “International Symposium on Polyelectrolytes (2006)”. * To whom correspondence should be addressed. Phone: +49 331 5679447. Fax: +49 331 5679202. E-mail:
[email protected]. ‡ Max Planck Institute of Colloids and Interfaces. § Institut de Chimie Se ´ parative de Marcoule. | Laboratoire Claude Fre ´ jacques. ⊥ Queen Mary University of London. # Georg-August-Universita ¨ t.
imaging method for life, materials, and environmental sciences.10-13 At this stage, the resolution of the leading X-ray microscopy facilities is better than 20 nm and one may safely expect it to be soon below 10 nm.14,15 For wet carbonaceous samples, soft X-rays are optimal since in the so-called “water window” (284543 eV) the absorption by oxygen is sufficiently low that they can penetrate a 10 µm water film. For a transmission measurement based on absorption, the contrast is then given by absorption because of carbon. In the example we present below, we basically measure the carbon distribution with a resolution of about 20 nm. Polyelectrolyte multilayers have found widespread interest because of their ease of fabrication, versatility, and modularity.16 They have escaped all attempts of theoretical description, because (probably with exception of highly hydrated systems) they are nonequilibrium structures. Hence, also all properties are given by the preparation history and thus deviate nominally for the same system with minute changes of their preparation. This situation may change now since it has been shown that for many systems there is a glass transition not too far above room temperature,17,18 and the transition temperature can also be tuned via pH and salt.19,20 Thus, it is possible to transform a film into the molten state and then return to the glass at wellreproducible conditions. By colloidal probe force spectroscopy, it could be shown that the elastic modulus differs by 2 orders of magnitude between these two states.18 By means of confocal fluorescence microscopy with polyelectrolyte capsules, which may be considered as free-standing multilayers, it was shown that in the glassy state the permeability is drastically reduced.21,22 This indicates that the wall has been densified, and we will show below how transmission X-ray microscopy (TXM) can be used to quantify this feature. With this example, we demonstrate the potential of the technique at the present stage and at the end we will sketch the limits and perspectives of the technique for the next decade. Materials and Methods Materials. Poly(styrenesulfonate sodium salt) (PSS, Mw ) 70 kDa), poly(allylamine hydrochloride) (PAH, Mw ) 70 kDa),
10.1021/jp070360+ CCC: $37.00 © 2007 American Chemical Society Published on Web 04/12/2007
Soft X-ray Microscopy To Image Polyelectrolytes
J. Phys. Chem. B, Vol. 111, No. 29, 2007 8389
Figure 1. Scheme of the transmission X-ray microscope at BESSY.
and poly(diallyldimethylammonium chloride) (PDADMAC, Mw ) 200-350 kDa) were purchased from Aldrich (United States). Sodium chloride, hydrofluoric acid (40%), ammonia (25%), and hydrogen peroxide (30%) were purchased from Fluka (Switzerland). Hydrochloric acid (1 M) was obtained from Merck (Germany). Ammonium fluoride was purchased from Riedelde-Hae¨n (Germany). All chemicals were used without further purification except for PSS, which was dialyzed against Milli-Q water (Mw cutoff 12.4 kDa) and was lyophilized. Solutions were prepared freshly prior to use. Monodisperse silica particles with a diameter of 4.5 µm were obtained from Microparticles GmbH (Germany). The water used in all experiments was prepared in a three-stage Millipore Milli-Q Plus 185 purification system (Millipore, United States) and had a resistivity higher than 18 MΩ·cm. Capsule Preparation. Hollow polyelectrolyte capsules were fabricated using the layer-by-layer (LbL) technique as described previously.5,23 Prior to the deposition of polyelectrolytes, silica particles were washed three times with water. For the preparation of PAH/ PSS capsules, the surface of the silica templates was etched in a mixture of 25% NH3:30% H2O2:H2O (1:1:5) for 20 min (25 mg‚mL-1 SiO2) to ensure a better attachment of the polymers to the bare SiO2 surface. Thereafter, the particles were thoroughly washed with water. The alternating adsorption of polyelectrolytes onto the surface of the silica particles was carried out from 2 mg‚mL-1 polymer solutions containing 0.5 M NaCl, starting with the positively charged polyion since the silica particles are negatively charged within the used pH range of 4-6. After 15 min of adsorption, the supernatant was removed by centrifugation at 1500g, and the particles were washed three times with water to remove nonadsorbed polyelectrolyte molecules. The coating of silica cores with PAH/PSS requires special conditions: After etching of the cores, the pH of the silica suspension in water (40 mg‚mL-1) was adjusted to 5 and the same volume of a 4 mg‚mL-1 PAH solution in 1 M NaCl pH 5 was added for the deposition of the first layer. The following washing and adsorption steps were performed as described above but at pH 5. Since PAH forms a nonsoluble complex with SiF62- ions, it is not possible to dissolve coated particles with PAH as outermost layer without aggregation. Therefore, capsules with PAH at the outer surface were prepared by coating capsules after core dissolution with the polycation in 0.5 M NaCl at pH 5 as follows: 5 mL of a solution containing 2 mg‚mL-1 of PAH in 0.5 M NaCl was added slowly to 1 mL suspension of capsules (≈107 capsules/mL) while stirring in an ultrafiltration cell. After 15 min of incubation, the excess polyelectrolyte was removed by rinsing at least six times with pure water. After deposition of the desired number of layers, the silica cores were dissolved either in 0.1 M hydrofluoric acid (for
PDADMAC/PSS) or in 0.2 M ammonium fluoride buffer pH 4 (for PAH/PSS). The coated particles were added dropwise to the respective fluoride solution and were stirred for 40 min. Afterward, the resulting hollow capsules were washed again twice with the same fluoride solution for 20 min to remove the remaining SiF62- ions. Thereafter, the hollow shells were thoroughly rinsed with water until the pH reached a value of 6 and all fluoride ions were washed off. Temperature Treatment of Capsules. A 1.5 mL Eppendorf tube containing 100 µL of the aqueous capsule suspension was incubated at the respective temperature and time in a waterfilled Lauda Ecoline RE 112 thermostat (Lauda Dr. R. Wobser & Co. KG, Germany). Encapsulation. The encapsulation of dextran was done via heat-shrinkage:21 The (PDADMAC/PSS)4 capsule suspension and a solution of 2 mg‚mL-1 FITC-dextran 10 kDa were mixed 1:1. After 1 h of equilibration, the mixture (100-200 µL) was heated for 20 min at 52 °C to shrink the capsules and thus to densify the shells. The shrunk capsules were then stored at room temperature for around 30 min to cool down before they were washed thoroughly (at least five times) with water to remove all surrounding molecules that were not encapsulated. The washing was performed using centrifugation at 2500g for 10 min. Transmission X-ray Microscopy (TXM). TXM measurements were conducted at the undulator U-41 at BESSY II (Berlin) using a wavelength of 2.4 nm (524 eV). The optical setup of the full-field transmission X-ray microscope is shown in Figure 1.24 Plane mirrors are used to direct the beam from the undulator into the TXM beamline. An off-axis transmission zone plate creates a demagnified monochromatic image of the undulator source at the sample position via a system of three mirrors. Rotation of the last two mirrors around the optical axis generates a hollow cone illumination to match the aperture of the objective to that of the condenser. The specimen is located in an environmental chamber in air, separated from the vacuum tube by outlet and inlet windows made of 180 nm thick silicon foil (Figure 2). A microzone plate (25 nm outermost zone width) behind the sample used as objective forms a magnified image on the CCD detector (1340 pixel × 1300 pixel; sensitivity of 57 counts per photon at 524 eV). A spatial frequency analysis (Fourier analysis) on an image of a test grating yielded a cutoff frequency corresponding to smallest resolvable features of about 20 nm for the used TXM setup. For measurements, 2 µL of capsule suspension was placed into the environmental chamber (Figure 2). The maximal thickness of the specimen between the polyimide foils is 10 µm but can be reduced by manual aspiration and a resulting slight bending of the foils into the chamber interior, which ensures adhesion between the two parts of the chamber. To
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Ko¨hler et al. value of the background noise is needed to convert the initially derived capsule profiles into transmission profiles, which are required for a quantitative evaluation. The transmission T denotes the radial intensity of a capsule I divided by the average intensity of the water background I0 around the shell:
T)
Figure 2. Environmental sample chamber for transmission X-ray microscopy.
I I0
(1)
This normalization yields therefore directly the transmission data of the pure polymeric material. The intensity depends on both the beam intensity and the local specimen thickness. For each capsule, we considered the background as the zone starting from the external radius up to 500 nm further outside; in this region, the specimen thickness can be considered as constant. The absorption of monochromatic X-rays by matter follows the Lambert-Beer law:25
T ) e-µ(X,E)‚F‚D
Figure 3. TXM micrographs of a temperature-shrunk (PDADMAC/ PSS)4 capsule recorded with different exposure times. The geometrical parameters of the shells obtained by quantitative analysis prove the increasing degradation of the polyelectrolyte network at long exposure times.
prevent a deformation of capsules, 4.5 µm silica particles were introduced as spacers into the sample solutions maintaining the windows at a respectable distance from each other. Images of capsules were acquired at room temperature with an exposure time of 3 s. This time turned out to be the optimum for capsule observation, where no significant radiation damage occurred, but with still a high-image quality. Figure 3 illustrates TXM micrographs of the same capsule recorded with three different exposure times, namely, 1, 3, and 12 s: The signalto-noise ratio increases with increasing X-ray exposure time. However, a quantitative image analysis (see below) revealed that at higher exposure times (>5 s) the external capsule radius Re slightly decreases, whereas the internal radius Ri increases. The resulting decrease in membrane thickness (h ) Re - Ri) can be attributed to radiation damages; the intense X-ray beam leads to a partial destruction of the polymeric network starting from the polymer/water interfaces (both internal and external). Moreover, the transmission profiles of such long irradiated samples show significant deviations from the used model, as the matter repartition within the membrane is not constant anymore. Therefore, 3 s was chosen as an optimal exposure time, the best balance between signal-to-noise ratio and radiation damage. Taking into account that the photon flux at the specimen is 3‚108 photons‚s-1·µm-2, this yields a photon density of 9‚108 photons·µm-2 (about 108 Gy).15,24 To keep the radiation damage as low as possible, an exposure time of 0.5 s was used for focusing. Quantitative Analysis of the TXM Micrographs. From the raw images, radial profiles of the capsules were extracted using the Image J software. The ordinate values of these plots represent the amount of light (photon density L) received by the CCD camera after passing through the specimen. For each capsule, conversion into intensity (photon flux) was then realized, using micrographs recorded at different exposure times (
