New Insight into Microgel-Stabilized Emulsions Using Transmission X

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New insight into microgel-stabilized emulsions using transmission x-ray microscopy: non-uniform deformation and arrangement of microgels at liquid interfaces Karen Geisel, Katja Henzler, Peter Guttmann, and Walter Richtering Langmuir, Just Accepted Manuscript • DOI: 10.1021/la503959n • Publication Date (Web): 12 Dec 2014 Downloaded from http://pubs.acs.org on December 14, 2014

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New insight into microgel-stabilized emulsions using transmission x-ray microscopy: non-uniform deformation and arrangement of microgels at liquid interfaces Karen Geisela*, Katja Henzlerb, Peter Guttmannb, Walter Richteringa* a

b

Institute of Physical Chemistry, RWTH Aachen University, Aachen, Germany

Helmholtz-Zentrum Berlin für Materialen und Energie GmbH, Institute for Soft Matter and Functional Materials, Berlin, Germany

Microgel-covered interfaces, e.g. in emulsions, attract much interest lately. Different imaging techniques have been used to image these interfaces, either flat or curved, to investigate their properties and appearance. Techniques like cryogenic-scanning electron microscopy (cryo-SEM) and confocal microscopy have provided valuable insight into microgel-covered systems but still have some disadvantages like part of the microgels being trapped in vitrified liquid or the need of fluorescence markers. Some of these disadvantages can be overcome by using transmission x-ray microscopy (TXM) which has the advantage of allowing the investigation of adsorbed and free microgels simultaneously. We used TXM to acquire tomographic image series of microgelcovered droplets and calculated 3D reconstructions from these image stacks. As a result, we

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could show that microgels deform anisotropically and penetrate into the oil droplets in the hydrated state. Additionally, 3D reconstruction gives an idea of the arrangement of microgels adsorbed to oil droplets and reveals that droplet stabilization is possible without full coverage of the interface with polymer segments.

Introduction Microgels are soft, polymeric particles that assemble at liquid interfaces and lower the interfacial tension very effectively1-5. They can thus be used to stabilize emulsions.6-13 Microgels composed

of

poly(N-isopropylacrylamide)

possess

special

properties

(e.g.

softness,

deformability and stimuli-sensitivity towards external parameters) that differentiate the respective emulsions from the ones stabilized with solid particles.8,

11, 14-17

In particular, these

emulsions can be broken on purpose by changing external parameters like temperature or pH, offering various applications for instance in biocatalysis.18-19 To get an extensive view on possible applications of microgel-stabilized emulsions, it is crucial to investigate the microgelcovered interface in detail. This has been done at flat and curved interfaces by using different cryo-SEM methods.20-28 These methods combine relatively easy sample preparation and experimental setup with the possibility to image deformation, arrangement and, to some extent, 3D location of the microgels at the interface. However, the determination of the exact size, shape and location at the interface is difficult because a large part of the microgels is surrounded by vitrified liquid. Although special methods like freeze-fracture shadow-casting (FreSCa) cryoSEM provide information about the protrusion height into the oil phase,29-30 the microgels’ shape in the water phase remains unseen.


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Another valuable method to image microgel-covered interfaces is confocal microscopy.31-34 However, this technique is limited in the sense that, similar to electron microscopy, no direct visualization of the microgels’ appearance is possible. Transmission x-ray microscopy (TXM) has been proven to be a useful tool for the analysis of biological material and gives direct insight into the internal structure of the species under investigation.35-37 The setup provided at the beamline U41-TXM at the Helmholtz-Zentrum Berlin - Electron Storage Ring BESSY II allows acquiring a series of images taken under different viewing angles. Subsequently, sectional views can be reconstructed from these tilt series and a complete 3D-view of the sample can be gained. The investigation of biological material with x-rays is advantageous due to the large difference in x-ray transmission between carbon and oxygen at an appropriate wavelength (within the so-called water window between 4.37 nm and 2.29 nm).37 Samples with a thickness of several µm can be studied with TXM, contrary to the small sample thickness in e.g. cryo-transmission electron microscopy (TEM). The possibility to image relatively thick samples is one of the main advantages concerning the investigation of microgel-stabilized emulsions with a droplet diameter of several µm. Previous projects investigated large droplets with diameters a lot larger than the microgel diameter. Here we investigate smaller droplets where the size of the droplets is in the range of the microgels due to sample preparation requirements for TXM. Due to sample preparation (cp. Methods section), the investigated droplets in TXM are not much larger than the microgels attached to theses droplets. Furthermore, TXM opens the possibility to investigate adsorbed and bulk microgels in an aqueous environment and in 3D simultaneously, making this method superior to e.g. cryoSEM methods. Still, the remaining difficulty in TXM-imaging of microgels lies in their hydrated state, resulting in a low contrast between microgels and the surrounding water. Thus, contrary to


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emulsions stabilized by solid particles (Pickering emulsions), where imaging of particle-covered droplets is possible even with light microscopes due to large particle size and high contrast, 14, 16, 38-41

imaging of microgel-stabilized emulsions is a challenge.

In this contribution, we present tilt series and 3D reconstructions of microgel-covered emulsion droplets. A special, non-uniform deformation of the microgels at the droplets is visualized where we give proof that the microgels protrude into the oil phase in the hydrated state. Furthermore, a 3D impression of the microgel arrangement around droplets is gained.

Results The synthesis and characterization of the microgels used in this study are described in detail elsewhere.23 Microgels were synthesized by surfactant free precipitation polymerization following a semi-batch synthesis approach, leading to core-shell microgels with inhomogeneous charge distribution but rather homogeneous crosslink density. The core of the microgels consists of poly(N-isopropylacrylamide-co-methacrylic acid), P(NiPAm-co-MAA), while the shell does not contain any MAA but only P(NiPAm). Thus, a deprotonation of the acidic groups at high pH induces charges that are mainly located in the interior of the microgel. The electrophoretic mobility is -0.5·10-8 m2/V·s at pH 3 and -1.24·10-8 m2/V·s at pH 9.23 Despite the low amount of surface charges, the microgel swells due to increased osmotic pressure and the hydrodynamic radius increases from 610 nm at pH 3 to 785 nm at pH 9. Even though the charges in the core are shielded by an uncharged shell, the size and thus properties like deformability and softness change with changing pH. The resulting properties at the interface may also be different and not directly influenced by coulomb interactions.32, 42


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TXM images of microgel-stabilized emulsions were taken in the cryo-state at different viewing angles. Due to the fact that x-ray radiation can produce beam damage in organic samples, images at 0° tilt angle were taken before and after acquiring tilt series to check the samples for radiationinduced changes. Neither the microgels nor the droplets suffered from beam damage (Figure S1). The dimensions of the bright concave areas at the droplet surface were also not affected by the xray beam and were therefore attributed to hydrated microgels protruding into the oil droplet, as discussed in more detail below. Additionally, the droplets’ internal structure does not change during tomogram acquisition which excludes beam damage in this case. TXM apparently reveals inhomogeneity inside the oil droplets that differ from the expected homogeneous oil phase and more sophisticated methods are required to shed light on the origin and details of the droplet structure. As we cannot explain these structures sufficiently within the scope of this contribution, no further analysis is presented. Images taken at 0° tilt angle are presented in Figure 1. The oil droplets appear black resulting from significantly lower transmittance than the water phase and the microgels, which are located at the oil-water interface around the droplets. Additionally, free microgels are still present in the aqueous phase due to an excess of microgels during emulsion preparation. Nevertheless, microgels are accumulated around the droplets demonstrating their high affinity to interfaces, as it has been reported before by several groups using different imaging techniques and measurements of the interfacial tension. The porous, sponge-like structure of the microgels is also visible in some cases (Figure 1c, higher magnification image in Figure S2). This characteristic microgel structure has, to the best of our knowledge, only once been imaged before at the surface of emulsion droplets with microscopic methods.23


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Figure 1. TXM images of microgel-stabilized emulsions taken at 0° tilt angle. The droplets appear as spheres of low transmittance. The microgels are either arranged around the droplets or free in the water phase. a, b) charged microgel at pH 9, heptane as oil phase. c, d) uncharged microgel at pH 3, decane as oil phase. The adsorbed microgels appear as round objects with a diameter of 1.33 ± 0.07 µm at pH 9 (Figure 1 a and b) and 1.25±0.06 µm at pH 3 (Figure 1 c and d). The difference in size between charged and uncharged microgels is thus very small even though the size in bulk depends on the pH.23 This is not surprising because in previous studies it was shown that the microgel behavior and the degree of spreading or deformation at the interface are not influenced by the presence of charges in a straightforward manner even if the charges are present at the surface of the microgels.26, 32, 42 Additionally, the microgel size at the droplet surface is just slightly larger than the diameter of the non-adsorbed microgels (d = 1.30±0.04 µm at pH 9 and d = 1.17±0.06 µm at pH 3), indicating that the microgels experience little deformation. This is in contrast to other studies


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concerning microgels at liquid interfaces where a core-corona morphology with a dense core surrounded by a corona of flat, spread polymer segments was found to cover the interface in close hexagonal packing.22, 26 However, these studies were performed on microgels smaller than the ones used in the TXM measurements, and size may influence the microgels’ deformability at the interface (see Discussion). Exemplary views from different angles of one of the tilt series are shown in Figure S3. Full animated videos of the tilting can also be found in the Supporting Information. Tomograms were calculated from the tilt series using TomoJ,43-44 a plugin for the publically available software ImageJ.45 Videos of sample tomograms are given in the Supporting Information. One aspect of a tomogram is shown in Figure 2 in more detail: features at the droplet surface indicate that the microgels penetrate into the droplets. This supports the assumption that the main part of the hydrophilic microgels is hydrated and situated in the water phase. Due to the very low solubility of microgel particles in oil, only a small but still hydrated part is situated in the oil phase.26,

46

Thus, small concave areas at the surface of the droplets represent the microgels’

penetration into the oil.

Figure 2. Z-view through deformed microgels at the surface of an oil droplet. The microgels’ deformation and penetration into the droplet can be seen in d (white arrows). Images are cut from


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the tomogram reconstructed from the tilt series corresponding to figure 1d (videos S3 and S4). The distance in z-direction between each image is 0.25 µm. The deformation seems to be unaffected by charges in the microgels as the ratio of short and long axis of the deformed microgels is around 0.85 for the charged as well as for the uncharged microgel. Although microgels undergo pH-dependent swelling in bulk and thus possess different deformability, the potentially resulting difference in the deformation at the interface cannot be resolved with TXM in this case. It was already shown earlier that the microgel deformation at flat oil-water interfaces does not depend on the presence of charges in the microgel.26 The deformation of the microgel is thus independent from the swelling behavior arising from charge effects. Assuming that the microgels penetrate into an otherwise spherical droplet, the approximate penetration depth is around 300 nm. This corresponds to 20% (pH 9) and 30% (pH 3) of the size in bulk, as measured with dynamic light scattering (DLS) and published previously.23 This percentage is in the same range as the protrusion height determined in another study for smaller microgels.26 It was possible to calculate a 3D model from the tomograms by using the software imod.47-48 The model represents microgels as spherical objects arranged around a spherical droplet (Figure 3). The 3D representation illustrates the centers of mass and gives an impression of the position of the microgels at the droplet surface. As discussed earlier, spherical symmetry is a harsh simplification of the real appearance of both microgels and droplets. The microgels arrange randomly around the droplet with large uncovered areas between them. This is in contrast to previous publications, where hexagonal arrangement was found for large arrays of microgels at flat and curved interfaces.1,

20, 22-23, 26, 33-34, 49-56

However, the sample


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preparation and experimental setup in TXM only allows to image small droplets and the resulting consequences on microgel arrangement are discussed in detail in the following section.

Figure 3. Snapshots of a 3D illustration of microgels (green) around an oil droplet (black). A 360° view is provided as video in the Supporting Information (S5). The scale bar is 1 µm. Discussion TXM imaging showed that microgels indeed accumulate around oil droplets and an excess of microgels exists in the bulk phase. The main part of the microgels at the droplet surface is situated in the water phase and only a small, hydrated part protrudes into the oil phase, similar to what has been proposed previously.26, 34 However, there are some differences between the results presented above and previously published studies on microgel-covered liquid interfaces. First, the deformation of the microgels


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is different from the one observed in previous measurements, including the absence of spreading at the interface and the resulting similar size of free and adsorbed microgels. Second, the arrangement is different from that observed on large droplets and flat interfaces. In the following paragraph, these aspects will be discussed. It is possible to investigate microgels adsorbed at droplet surfaces as well as free microgels simultaneously with TXM. Previous publications showed either microgels at interfaces or microgels in bulk, but especially the extensively used cryo-SEM methods are restricted to imaging the interface. Microgels in bulk have a spherical shape and, surprisingly, TXM imaging revealed that this almost holds for adsorbed microgels, too. Some of the adsorbed microgels protrude into the oil droplet and are thus deformed, but otherwise these microgels are spherical. It could have been anticipated that the microgels spread at the interface to cover a large area with polymer segments and the absence of spreading may be due to different reasons. First, the crosslinker distribution and crosslink density have strong influence on the deformability of microgels. The crosslinker distribution in conventionally synthesized microgels is not homogeneous but decreases towards the periphery of the microgel.57 A semi-batch method was used for the synthesis of the microgels in this study, leading to a homogenous crosslinker distribution that may hinder spreading due to the crosslinked shell. However, cryo-SEM images of emulsion drops show that the same microgels are connected with filaments resulting from spreading of the polymer shell23 and homogenously crosslinked microgels have also been shown to spread at planar interfaces.26 These equivocal findings lead to the conclusion that there must be other properties inhibiting spreading than the crosslink density. Additionally, the low contrast between polymer and water may also cause the filaments to remain unseen in TXM if the spreading was small.


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TXM allows investigation of droplets with a diameter of several µm but the droplet size distribution of the used emulsion is very high, ranging from several µm to hundreds of µm. The blotting of the emulsion on the grid results in removal of large droplets and thus only small droplets remain where the droplet size is not much larger than the microgels’ size (cp. Figure 1). Small emulsion droplets imply that the microgels virtually see a bent interface which may also constrict spreading. Microgels adsorbed to large drops do not experience spreading either but a combination of large microgel size and curved interfaces may be energetically unfavorable and thus prevent the microgels from spreading. High energy input is needed to deform large particles at the interface, or, in other words, the energy that is gained if the particles spread at the interface and cover a high area with polymer segments is smaller than the energy that is necessary to produce the spreading. If the extend of spreading was small in comparison with the overall microgel size, it could also be assumed that the spreading cannot be resolved due to the large size of the microgels. Additionally, the contrast between microgels and water is naturally low and complicates an analysis of the exact appearance of the microgels due to blurred transitions from microgel to surrounding water. The low contrast was also an issue when droplets covered with microgels with a hydrodynamic radius of 100 nm or 300 nm, respectively, were imaged. The microgels were not visible due to their small size and the low contrast between the microgels and the surrounding water. It was thus not possible to analyze the deformation or arrangement of small microgels. The 3D representation revealed that microgels arrange randomly around droplets with distances larger than the microgel diameter, i.e. they are not closely packed or otherwise in contact (Figure 3). No hexagonal packing is observed, contrary to previous experiments performed on flat interfaces and large emulsion droplets. Due to residues of the initiator, the


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microgels have a small negative surface charge even at low pH when the acidic groups are protonated. At high pH, deprotonation leads to an increase in surface charge, even though the charges in the core are still shielded by the uncharged shell (the electrophoretic mobility of the microgels is given in the results section).23 It was shown in previous studies that electrostatic repulsion does not govern the arrangement at microgels at oil-water interfaces.56, 58 Additionally, experiments performed on flat microgel-covered interfaces in a Langmuir trough indicate that charged microgels can be compressed even further than uncharged ones before changes in the surface pressure can be measured.42 Therefore, it is assumed that electrostatics do not dominate the microgel arrangement at droplets neither at low nor at high pH. Microgels adsorbed to an oil-water interface are fully hydrated but nevertheless partially situated in the oil phase. The interface is bent around the hydrated particles and the microgels are not in direct contact with the oil phase; thus, the collapse of the microgels is avoided (Figure 2) Nevertheless, the dangling chains at the surface of the microgels are in contact with the oil phase and we expect these chains to be collapsed. However, this cannot be visualized with TXM. It was also shown by pendant drop experiments that the VPTT of PNiPAm microgels does not change at the alkane-water interface, even though the dangling polymer chains may be collapsed when they are in contact with the oil phase.2,

4

The resulting deformation of the oil-water

interface is much larger and thus energetically unfavorable in the case of large particles, leading to a reduced coverage of the interface as compared to smaller microgels. In general, the arrangement at small drops may well differ from large and flat interfaces due to the already mentioned size and curvature effects. Furthermore, the adsorption from the bulk, as it is the case in emulsion preparation, cannot be controlled accurately and different microgel structures and morphologies have also been found previously that deviate from hexagonal arrangement.46,

56


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These results show that stable emulsion droplets can be produced without the interface being covered completely with polymer segments. The solubility of the microgels in the oil phase may also have effects on the behavior of microgels at the interface. In this study, heptane and decane were used which are non-solvents for PNiPAm microgels and do not swell the microgels. A distortion of the oil-water interface is observed, as discussed earlier. Previous studies on microgel-stabilized water-in-octanol emulsions revealed pronounced differences to emulsions containing alkanes as oil phase.23-24 PNiPAm microgels can be swollen by octanol, even though the solubility is still low, and they change their swelling properties in bulk after contact with octanol. They lose, for instance, their temperature-responsiveness and the resulting emulsions are of the water-in-oil type although the microgels are dispersed in the water phase. This represents anti-Finkle behavior.23-24 Water and octanol are also soluble to a certain extent and there is an interfacial region of saturated solvents that has effects on the properties of the microgels present at that interface. They are at least partially swollen by octanol and it is thus assumed that the interface does not bend around the microgels as it is the case when alkanes are used as oil phase. As a result, the deformation of microgels at octanol-water interfaces would probably be different from alkane-water interfaces. The use of collapsed microgels is another possibility to investigate the influence of the microgels’ deformability and swelling properties on their deformation and arrangement at the interface. Different experiments can be conducted. First, sample preparation can be performed at temperatures above the microgels’ volume phase transition temperature (VPTT). PNiPAm-based microgels possess a VPTT of 32°C, meaning that sample preparation has to be performed with external temperature control. Additionally, the plunge-freezing of a sample at elevated temperatures requires a special setup. Secondly, PNiPAm microgels collapse in solvents


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containing water and methanol in a certain ratio, which is known as the cononsolvency effect. If the aqueous phase contains 20 mol% methanol, microgels collapse and adsorb to the interface in the collapsed state without the need to increase the temperature.59 Thirdly, equimolar copolymers of NiPAm and DEAm (N,N-diethylacrylamide) possess a VPTT in water of 20°C.60 This also allows the preparation of interfaces covered with microgels in the collapsed state at room temperature. It has already been shown that the coverage of the emulsion droplets depends on the preparation temperature of the emulsion,25 and we expect that collapsed microgels adsorb to the interface in a different state than the swollen ones. However, the experiments mentioned above are experimentally challenging and beyond the scope of this paper. They will be pursued in future projects and can provide important information about the effects of deformability and microgel softness. It would then be possible to compare interfaces covered with swollen or collapsed microgels directly.

Conclusion TXM tomography is a valuable tool to investigate microgel-stabilized emulsions in-situ and to receive a 3D image of the system and the appearance of both droplets and microgels. Direct and simultaneous imaging of microgels at droplet surfaces and in bulk provides valuable information about the porous structure of microgels and the deformed part of the microgels protruding into the oil phase in the hydrated state. Furthermore, tomograms and 3D visualization of microgelcovered droplets give new insight into the microgel arrangement with the result that, contrary to previous findings, it is not necessary to cover the interface completely with polymer segments to achieve stable droplets. This combination of information on different scales, namely the overall appearance of microgel-covered droplets and the internal structure of the microgels, is an


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important advantage of TXM over other microscopic methods like electron microscopy or confocal microscopy.

Experimental Microgel synthesis and characterization. Microgel synthesis and characterization was described in detail elsewhere.23 In brief, a degassed solution of NiPAm, MAA and N,N’methylene-bis-acrylamide (BIS) as crosslinker was heated to 70°C and the polymerization was initiated with potassium persulfate (KPS). 15 min after the first turbidity was observed, further NiPAm and BIS was added continuously with a syringe pump. After the addition was complete, a second portion of KPS was added and the reaction was left to proceed for 5 h. This two-step procedure leads to microgels with more homogeneous swelling properties than conventional precipitation polymerization. Different reaction rates of monomers and crosslinker do not lead to an inhomogeneous crosslinker distribution due to the addition of reactants in the course of the synthesis

Emulsion preparation. Emulsions were prepared from 4.2 mL microgel dispersion in water (1 wt%) at pH 3 or pH 9 and 1.8 mL n-heptane or n-decane. Microgel dispersions of samples 1a and 1b contained 16 mM CsCl to increase the contrast. Changes in the alkane chain length of the oil do not influence the resulting emulsion. Emulsification was performed at room temperature with an Ultra-Turrax T25 homogenizer (Janke and Kunkel IKA-Labortechnik) at 8000 rpm for 30 s.

Transmission x-ray microscopy. Specifically designed gold grids (HZB-2) coated with perforated carbon foil (R2/2) were purchased from Quantifoil Micro Tools GmbH, Jena,


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Germany. A detailed description of the grids can be found elsewhere.36 After plasma treatment for hydrophilization, the grids were mounted in a plunge freezer. The emulsion (5 µL) was applied to the grid (additional 5 µL of Au-NP solution (200 nm) were placed on the grid after the emulsion in the case of samples 1c and 1d) and was blotted with filter paper. The sample was then vitrified by plunge-freezing in liquid ethane35 and stored in liquid nitrogen afterwards. TXM measurements were performed at the beamline U41-TXM at the Helmholtz-Zentrum Berlin – Electron Storage Ring BESSY II with a 25 nm zone plate objective and a photon energy of 510 eV. Tilt series were recorded with a step size of 1° from -60° to +60° (sample a), -50° to +50° (samples b and c) or -65° to +65° (sample d). Special care was taken that no features like the grid bar moved into the field of view during sample rotation.

Image analysis. Image analysis and measurements of microgel size were performed with ImageJ45. Reconstructions from the tilt series (stacks with 101, 121 or 131 images, depending on the sample) were calculated with TomoJ,43-44 a plugin publically available for ImageJ. TomoJ allows fiducialless alignment by creating landmarks from image features and following these landmarks during alignment and reconstruction. Reconstruction was performed using the SIRT algorithm with 30 iterations and a relaxation coefficient of 1. The thickness of the reconstruction was chosen according to sample conditions. The 3D model was reconstructed using imod47-48 and the drawing tool and interpolation plugins. The sculpture tool was used to draw a circle around the microgel (or droplet) at the respective slide with the correct diameter previously measured with ImageJ. The circle was then automatically extended in z and –z direction assuming a spherical appearance of the feature.


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ASSOCIATED CONTENT Supporting Information. Images of oil droplets before and after tomogram recording; illustration of porous, sponge-like structure of microgels; example images of a tilt series of microgel-covered droplets; videos of tilt series and reconstructions of microgel-covered droplets; video of 3D arrangement of microgel-covered droplet. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *[email protected] *[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT We thank Basel Tarek, Sergey Kapishnikov and Ruben Cardenes for advice concerning tomogram processing and reconstruction. We thank the Helmholtz-Zentrum Berlin – Electron Storage Ring BESSY II for the allocation of beamtime at the beamline U41-TXM and for financial

support.

KG

and

WR

acknowledge

financial

support

of

the

Deutsche

Forschungsgemeinschaft and the Sonderforschungsbereich 985 “Functional Microgels and Microgel Systems”.


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Cohin, Y.; Fisson, M.; Jourde, K.; Fuller, G. G.; Sanson, N.; Talini, L.; Monteux, C.,

Tracking the interfacial dynamics of PNiPAM soft microgel particles adsorbed at air-water interface and in thin liquid films. Rheol. Acta 2013, 52, 445-454. 4.

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New Insight into Microgel-Stabilized Emulsions Using Transmission X

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