Ptychographic X‑ray Tomography of Silk Fiber HydrationMorteza Esmaeili,†,∥ Jostein B. Fløystad,†,∥ Ana Diaz,‡ Kristin Høydalsvik,† Manuel Guizar-Sicairos,‡

Jens W. Andreasen,§ and Dag W. Breiby*,†

†Department of Physics, Norwegian University of Science and Technology (NTNU), Høgskoleringen 5, N-7491 Trondheim, Norway‡Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland§Imaging and Structural Analysis Programme, Department of Energy Conversion and Storage, Technical University of Denmark,Frederiksborgvej 399, DK-4000 Roskilde, Denmark

*S Supporting Information

ABSTRACT: Studying noninvasively the internal nanoporousstructure of a single Tussah silk fiber under different humidityconditions, we demonstrate for the first time the feasibility ofin-situ ptychographic tomography. The resulting 3D images ofthe silk fiber interior, obtained at both dry and humidconditions, yield quantitative information about the spatialdensity variations in the form of detailed maps of the size,shape, and orientation distributions of the nanopores insidethe silk fiber, revealing that the fiber swells anisotropically inhumid conditions, with the expansion taking place solelynormal to the fiber axis. Exploiting quantitative information on the fiber’s electron density, hydration was found to proceedthrough interaction with the silk protein rather than filling of pores.

1. INTRODUCTION

X-ray imaging has found broad applications in both life andmaterials science due to the highly penetrating and relativelygentle nature of X-ray interactions with matter. After Rontgen’sdiscovery in 1895, X-ray imaging was until the late 1990ssynonymous with absorption contrast, which has its physicalfoundation in the absorption’s dependence on the atomicnumber Z. More recently, several phase contrast imagingmethods have been established,1 yielding improved contrast forfundamental reasons described later in this article.Traditional X-ray microscopy has been hampered by the

absence of suitable X-ray objective lenses, which severely limitsthe resolution.2 Coherent diffractive imaging (CDI) is a familyof techniques based on evading the use of an objective lensaltogether, instead reconstructing the image from coherentscattering patterns by means of suitable algorithms andconstraints. The CDI technique of ptychography, as firstdemonstrated by Faulkner and Rodenburg,3 builds on ideas forphase retrieval by Hoppe.4,5 With ptychography, the field ofview is scanned by a coherent X-ray beam sufficiently small toallow Nyquist sampling in reciprocal space (up to a fewmicrometers in current experiments). While ensuring aconsiderable degree of spatial overlap of neighboring exposures,a series of coherent scattering patterns are recorded in the farfield using an area detector (cf. Figure 1). The overlap itself is acrucial concept for ptychography to work, as the redundantinformation created by overlapping illuminations of the objectis used as one of the two major constraints in the iterativereconstruction algorithm.6,7 The other crucial constraint is thatthe modeled far-field intensity distribution should match the

experimentally obtained scattering patterns. The phasereconstruction routine thus essentially consists of repeatedFourier transforms back and forth between the object plane andthe detector plane, while using these constraints to update thereconstruction estimate. The reconstruction yields both phaseand amplitude images with a reconstructed pixel sizedetermined by the wavelength and the highest accessiblescattering angle (typically tens of nanometers in currentexperiments). Based on the reconstructed 2D projectionimages from ptychography measurements obtained at differentsample rotation angles, full 3D tomography reconstructions canbe obtained,8 which is a highly desired input for many researchfields.9,10 A particularly appealing aspect of X-ray ptychographyis that it opens for a wide range of in-situ experiments involvingchallenging sample environments.Being electromagnetic waves, X-rays can be assigned an index

of refraction n, conventionally represented by n = 1 − δ + iβ.The real part δ is typically about 10−5 and is responsible forrefraction, while the imaginary part β ∼ 10−7 accounts forabsorption. That δ is at least an order of magnitude larger thanβ for hard X-rays, thus having a higher potential for contrast, isthe fundamental reason for the interest in X-ray phase contrastimaging.1 The refractive index n can (except near absorptionedges) readily be estimated to high accuracy from the massdensity and composition stoichiometry of the material understudy. In a ptychographic tomography experiment, the 3D

Received: October 9, 2012Revised: December 21, 2012Published: January 4, 2013

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distributions δ(r) are obtained, which are readily converted toelectron density distributions ne(r).

11 Furthermore, lengths mayalso be measured with high accuracy as the pixel size dependssolely on well-known experimental parameters.Silk is a natural protein fiber obtained from cocoons made by

silkworm larvae. Its properties and applications have led tomuch scientific interest in silk.12−20 The fibers are known topossess a rather faceted, often roughly triangular, externalshape, which by refraction gives silk its iridescent appearance. Itremains today a unique material for the textile industry becauseof its luxurious feel and look. It is furthermore hypoallergenic,highly water absorbent, robust, and strong. The main proteinsconstituting silk are fibroin, the structural center of the silk, andsericin, the “sticky” material surrounding the core which isremoved in the industrial process of degumming. The fibroin isa block copolymer consisting of hydrophobic crystalline β-sheets connected by short hydrophilic links, giving silk itsstrength and resilience.13 The water uptake, or hydration, isclosely related to the macromolecular structures in the fiber andhas been subjected to numerous scientific studies resulting in arather good understanding of the thermodynamical interplaybetween α and β protein structures, water, and temper-ature.19,20 The hydration studies have been based on techniquesranging from weight gain measurements,18 via Raman studies19

to wide- and small-angle X-ray scattering,14,19 Fourier transforminfrared spectroscopy (FTIR),20 and nuclear magnetic reso-nance (NMR).21 Despite the wide interest in the topic,methods for direct observation of the hydration process havenot been published.It is well-known that silk fibers possess nanopores and that

these pores affect the physical properties of the fibers.14,15 Therole the pores play in the water uptake process appears unclear,and one can speculate whether capillary action merely serves totransport humidity to drier regions inside the fiber or whetherthe pores also act as water reservoirs. Understanding thestructure of the pores, including their orientations, size, and

shape, is important also for understanding the performance ofsilk fibers in new and challenging technological applications likeorganic electronics for textiles22 and tissue engineering.17

Here, we present the first in-situ ptychographic tomographyexperiment, imaging a single Tussah (also known as Tasar) silkfiber under different humidity conditions. The porous structureof the silk fiber could be clearly resolved, and by comparing thetomograms obtained in the dry and humid state, we find thatthe water uptake leads to an anisotropic swelling of the fibercross section.

2. EXPERIMENTAL SECTIONMaterials. The degummed Tussah silk fiber was studied as received

from Aurora Silk.23 Tussah silk has a wedge-shaped cross section withtypical major axis of 25−45 μm.18

X-ray Ptychographic Tomography. The X-ray ptychographictomography experiment was carried out at the cSAXS beamline(X12SA at the Swiss Light Source) with 6.2 keV radiationcorresponding to an X-ray wavelength λ = 1.998 Å. The undulatorX-ray beam passed through a double-crystal Si(111) monochromatorbefore reaching a 2.5 μm pinhole, located 4.5 mm upstream of thesample position as illustrated in Figure 1. To control the humidityconditions, a custom sample cell was used, consisting of a base forfixing the fiber to the piezo-stage of the sample goniometer and a freelysuspended cap being stationary with respect to the pinhole and theincoming beam (see also Supporting Information, Figure S1). Thefiber was fixed at both ends without straining it. The cap was equippedwith temperature and humidity sensors and 200 nm thick Si3N4

membranes serving as windows for the incoming and scattered X-raybeams. Flushing the cap with dry or humidified N2 allowed the relativehumidity level to be controlled between ∼5 and ∼95%. We will referto 5% and 95% relative humidity as the “dry” and “humid” state,respectively. The sample assembly with the fiber mounted verticallywas attached to a 3D high-precision piezo positioning stage. Arotational stage collinear with the fiber axis, placed below the piezostages, was used for tomography. In total, 88 and 176 projections wererecorded in the dry and humid state, respectively, in both casescovering an angular range of 175°. For each chosen projection angle,the sample was scanned in the plane perpendicular to the incomingbeam, yielding 347 nonperiodically arranged exposures, covering arectangular field of view of 42 (horizontal) × 20 μm2 (vertical). Thescattering patterns were collected with an acquisition time of 0.2 susing a Pilatus 2M hybrid pixel detector24 located 7198 mmdownstream of the sample position behind a He-filled flight tube.Collecting one complete tomography data set with 181 projectionsthus lasted about 7 h. We used the data contained in a central area of192 × 192 pixels of the detector, equivalent to a wavevector transfer 0Å−1 < Q < 0.018 Å−1, as input for the ptychography reconstructionalgorithm.7 The postprocessing steps consisted of phase ramp removal,phase unwrapping, and projection alignment.25 The tomographyreconstruction was then performed using a modified filtered back-projection algorithm.

Wide-Angle X-ray Scattering. Wide-angle X-ray scattering(WAXS) patterns of a bundle of Tussah silk fibers were acquired atboth dry and humid conditions. The setup was configured with arotating anode source equipped with a Xenocs collimating multilayermirror emitting a monochromatic beam of Cu Kα radiation(wavelength λ = 1.54 Å) and a Pilatus 1M area detector. Fiberswere mounted vertically. The sample-to-detector distance was 150mm, and the exposure time was 3 h. The wavevector transfer wasdefined as Q = 4π sin θ/λ, where 2θ is the scattering angle.

Scanning Electron Microscopy. Scanning electron microscopy(SEM) on a single silk fiber was performed using a FEI HeliosNanoLab DualBeam (Focused Ion Beam (FIB)-SEM) instrument.The silk fiber was coated with gold and subsequently cut with a Ga ionbeam normal to the fiber axis, leaving the cross section exposed. Thiscross section was imaged by SEM. The tilt angle between the electron

Figure 1. Experimental setup employed for acquisition of ptycho-graphic tomography data at the cSAXS beamline (not to scale). Thehighly coherent incoming beam (a) passes through a 2.5 μm pinhole(b) and interacts with the sample situated inside a plastic cap forcontrolling the local environment (c) (see also SupportingInformation, Figure S1), which was mounted 4.5 mm downstreamfrom the pinhole. Scattering patterns are recorded by a 2D detector(d) located 7198 mm behind the sample position.

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and Ga ion beams was 52°, which has been corrected by rescaling theimage.

3. RESULTS AND DISCUSSION

Two full tomography data sets were measured on the same silkfiber in an N2 atmosphere, first with a relative humidity ofabout 5% (“dry” state) and then with about 95% relativehumidity (“humid” state), aiming to see if the presence ofhumidity changes the fiber morphology and dimensions.Figures 2a and 2b present two renderings of the reconstructed3D tomographic volume for the humid state. The porousstructure is readily seen, with elongated pores running parallelwith the fiber axis in the sample interior. The shading in the 3Drendering indicates the wedge-shaped cross section of the fiber,having a long axis of ∼26 μm and a short axis of ∼13 μm.Nanopores with diameters of a hundred to several hundrednanometers are clearly detectable and are seen to be orientedalong the main axis of the fiber. The nanopores are distributedinhomogeneously, with higher concentration at the corners ofthe fiber. More than 90% of the pores were found to extendthroughout the 13 μm long field of view, suggesting that theaverage pore is significantly longer than the field of view. Ahistogram of the pore cross section areas is shown in Figure 2c,indicating that for the pore sizes we can resolve observationfrequency increases with decreasing size. Complementary SEMimages (see Figure 2d) show that the silk fibers possess poreswith diameter smaller than 50 nm, which were not resolved inthe ptychography experiment.A necessary condition for comparing the tomography scans

acquired in different humidity conditions is to make sure thatthe two data sets are acquired in exactly the same region of thefiber. This was readily verified from the presence of severaleasily recognizable features of the pore structure, as exemplifiedin Figure 2a, in both 3D reconstructed volumes of the fiber (see

also the Supporting Information, Figure S3). The dimensionsof the fiber are clearly affected by the relative humidity level inthe surrounding atmosphere, exhibiting a marked swelling, asshown in Figure 3a. Moreover, the swelling is anisotropic: Theexpansion is 4−5% normal to the fiber axis, while we wereunable to identify any expansion along the fiber axis; if present,the expansion is less than 1%. This is supported by the fact thatthe fiber hardly moved along the fiber axis at all when changingto the humid state: As the length of the fiber is severalmillimeters, even a small relative expansion along the fiber axiswould result in a substantial and readily detectable shift.Measuring the volume inside the perimeter of the fiber in bothstates (thus including the volume of pores), we find that thevolume increases by 10.3 ± 1.0% in the humid state, agreeingwell with the linear expansions described above.It is interesting to note that all the major pores observed in

the fiber are seen in both the dry and humid state. Thisobservation is supported by the pore size distribution (cf.Figure 2c) remaining the same after introducing humidity. Ifthe pores would play an active role as water reservoirs, wewould have expected to observe filling of pores that wereoriginally empty. This would manifest itself by pores that arevisible in the dry state becoming nearly indistinguishable fromthe silk in the humid state, as the refractive index for silk isnearly equal to that of water. The observations of identical porestructures in both dry and humid states suggest that thehydration of the silk fiber proceeds by direct interaction withthe protein fibers, with the porous structure playing at most aminor role.The histograms of the voxel values in the reconstructed

volume shown in Figure 3b can be used for quantitative analysisof the density of the fiber. The leftmost peak corresponds tothe N2 surrounding the fiber and was centered at an electrondensity of ne = 0 e−/Å3, while the other peak at ne = 0.39 e−/Å3

Figure 2. (a) Slice of the silk fiber (humid state) along the fiber axis obtained by tomography reconstruction. Electron density has been mapped tocolor. The stripes observed are nanopores inside the silk fiber. The discontinuous pore is an example of the features used to confirm that the sameregion was imaged in both the dry and humid states. (b) 3D rendering of the porous structure inside of the fiber (humid state). The shadingindicates the rather elongated cross section of the fiber, having long axis 27.1 μm and short axis 13.3 μm. (c) Histogram of pore cross-section areas,indicating that pore sizes toward the lower limit set by the resolution of 0.02 μm2 are most abundant. The histogram was smoothed by kernel densityestimation;27 dotted lines indicate the uncertainty in the measurement. (d) Central part of a SEM image of a Tussah silk fiber cross section takenafter sectioning by a FIB, clearly showing that there are many small pores which are not detectable with the resolution of our ptychographyexperiment.

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represents voxels filled with silk and/or water. The finiteresolution in electron density explains both the unphysicalnegative tail and the fact that the contributions from water andsilk cannot be distinguished as the electron density for water isonly slightly lower than for silk (ne = 0.39 e−/Å3 for silk and ne= 0.33 e−/Å3 for water). Nevertheless, we obtain quantitativeinformation on the water gain through the total electron count,which is found by integrating the histogram. The total numberof electrons in the volume covered by the tomograms increasedfrom the dry to the humid state. Assuming that all gainedelectrons are bound to water molecules, we calculated the massof the absorbed water. Normalizing by the volume of the fiber,

we find that the fiber absorbed a substantial 0.11 mL of waterper mL of fiber.Two complementary and independent measurements of the

expansion of the fiber have been obtained: (i) the volumewithin the fiber perimeter and (ii) the volume of absorbedwater. We find that the above-mentioned 10.3% increase involume contained within the fiber perimeter (which includesthe volume of pores) is just large enough to accommodate thevolume of the absorbed water, keeping the density essentiallyconstant as observed experimentally. Thus, the pores need notstore water. As none of these measurements depend onresolving individual pores directly, we conclude that majorcontributions to water uptake and storage by pores, includingpores too small to resolve, is unlikely. Furthermore, the wide-angle X-ray scattering patterns in Figure 3c reveal that thecrystalline peaks of silk fibroin have not changed with thechange of relative humidity in the surrounding atmosphere,suggesting that absorbed water does not interact with thecrystalline regions of the fiber. Presumably, the water getsabsorbed in the hydrophilic amorphous regions known to existin silk.26

The refractive index obtained from both the humid and thedry measurements corresponds to an electron density of ne =0.39 e−/Å3. Knowing the approximate chemical composition ofthe protein-rich fiber, the mass density of the fiber is found tobe ρm = 1.23 g/cm3, which is about 6% lower than values (∼1.3g/cm3) previously reported with other techniques.18 Thediscrepancy between these two values could be due to thepresence of small unresolved pores in the sample, as found inSEM images of a similar silk fiber shown in Figure 2d. Themissing wedge due to the limited angular range of theexperiment was found not to influence the electron densityvalues, as demonstrated in simulated tomographic reconstruc-tions.A new approach to the scientific problem of imaging the

process of hydration has been presented, a topic of huge andgrowing importance in connection with e.g. microfluidics,28

here specifically applied to a silk fiber. We monitor non-destructively the hydration-induced swelling of the fiber in 3D.Obvious extensions of this work would be to study the effectsof other liquids and also to exert simultaneous mechanicalstress to the fiber. The ability of nondestructively and preciselymeasuring the electron density in 3D is key to these endeavors.As we have demonstrated, ptychography is a promising

approach for in-situ microscopy of delicate and/or buriedstructures. Efforts are currently being made to further improvethe resolution through both hardware29 and reconstructionalgorithm developments,30 and resolution better than 10 nmhas recently been demonstrated in 2D31,32 and down to 50 nmin 3D.29 The measurement time can potentially be reduced byan order of magnitude by (i) reducing the instrument dead-time and (ii) utilizing improved detectors with smaller pixelsthus allowing Nyquist sampling with bigger pinholes. From amaterials science point of view, X-ray ptychography is to befirmly established as a complementary method to transmissionelectron microscopy (TEM) and scanning electron microscopy(SEM), for which in-situ imaging is generally impractical.

4. CONCLUSIONSIn summary, we have presented an X-ray ptychographictomography study performed to characterize nondestructivelythe internal structure of an organic fiber, specifically Tussahsilk, under various humidity conditions. We demonstrate that

Figure 3. (a) Comparison of the fiber cross section in the dry (top)and humid (bottom) states. The atmosphere surrounding the fiber hasbeen removed for clarity. The major and minor axes of the fiberexpanded by 4% and 5%, respectively, when going from the dry to thehumid state. (b) Histogram of electron density for the exact samevolume in the dry and humid states. The increase of the peak at 0.39e−/Å3 at the expense of the peak at 0 e−/Å3 indicates a gain of volumewe attribute to absorption of water. A slight shift of the peak at 0.39e−/Å3 toward lower values is consistent with water absorption, aswater has a slightly lower electron density than silk. (c) Azimuthallyaveraged intensities of WAXS patterns of a bundle of Tussah silk fibersin both dry and humid states. The two sharp peaks at low Q valuesarise from the Kapton windows of the sample cell used.

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the nondestructive technique of ptychography can provide rich3D images of the internal porous structure of the silk fiber,confirming the existence of elongated nanopores directed alongthe fiber axis. It is clearly possible to extract detailedinformation on the disposition, size, and morphology of theinternal voids, in addition to quantitative measures of the localelectron density. In the current experiment, we used thisinformation to prove that silk fibers swell anisotropically whenexposed to humidity and that the hydration of the fiberproceeds through interaction with the protein matrix. Theabove-mentioned strengths of X-ray ptychography, togetherwith its comparably easy sample preparation and robustness,lead us to believe that X-ray ptychography will be a choice ofthe future as a powerful tool for in-situ study of nanostructuredmaterials.

■ ASSOCIATED CONTENT*S Supporting InformationSample cell details, alignment of projections for tomography,characteristic features used for alignment, and WAXS patternsof the fiber. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail dag.breiby@ntnu.no.Author Contributions∥These authors contributed equally.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors thank Ole Tore Buset and Xavier Donath fortechnical support and Andreas Menzel and Emil J. Samuelsenfor helpful comments improving the manuscript. We acknowl-edge financial support from The Research Council of Norwayand the industrial partners Statoil ASA, Total E&P Norge AS,Janus Holdings AS, Weenaas AS, and Swix Sport AS for partialfinancing of this work through the ColdWear project.

■ REFERENCES(1) Momose, A. Recent advances in X-ray phase imaging. Jpn. J. Appl.Phys. 2005, 44, 6355−6367.(2) Jacobsen, C.; Kirz, J.; Williams, S. Resolution in soft X-raymicroscopes. Ultramicroscopy 1992, 47 (1−3), 55−79.(3) Faulkner, H. M. L.; Rodenburg, J. M. Movable aperture lenslesstransmission microscopy: A novel phase retrieval algorithm. Phys. Rev.Lett. 2004, 93 (2), 023903.(4) Hoppe, W. Beugung im inhomogenen Primarstrahlwellenfeld. III.Amplituden- und Phasenbestimmung bei unperiodischen Objekten.Acta Crystallogr., Sect. A 1969, 25 (4), 508−514.(5) Hoppe, W. Beugung im inhomogenen Primarstrahlwellenfeld. I.Prinzip einer Phasenmessung von Elektronenbeungungsinterferenzen.Acta Crystallogr., Sect. A 1969, 25 (4), 495−501.(6) Bunk, O.; Dierolf, M.; Kynde, S.; Johnson, I.; Marti, O.; Pfeiffer,F. Influence of the overlap parameter on the convergence of theptychographical iterative engine. Ultramicroscopy 2008, 108 (5), 481−487.(7) Thibault, P.; Dierolf, M.; Bunk, O.; Menzel, A.; Pfeiffer, F. Proberetrieval in ptychographic coherent diffractive imaging. Ultramicroscopy2009, 109 (4), 338−343.(8) Dierolf, M.; Menzel, A.; Thibault, P.; Schneider, P.; Kewish, C.M.; Wepf, R.; Bunk, O.; Pfeiffer, F. Ptychographic X-ray computedtomography at the nanoscale. Nature 2010, 467 (7314), 436−439.

(9) Midgley, P. A.; Ward, E. P. W.; Hungría, A. B.; Thomas, J. M.Nanotomography in the chemical, biological and materials sciences.Chem. Soc. Rev. 2007, 36 (9), 1477−1477.(10) Mobus, G.; Inkson, B. J. Nanoscale tomography in materialsscience. Mater. Today 2007, 10 (12), 18−25.(11) Diaz, A.; Trtik, P.; Guizar-Sicairos, M.; Menzel, A.; Thibault, P.;Bunk, O. Quantitative X-ray phase nanotomography. Phys. Rev. B2012, 85 (2), 020104.(12) Marsh, R. E.; Corey, R. B.; Pauling, L. The structure of tussahsilk fibroin (with a note on the structure of beta-poly-L-alanine). ActaCrystallogr. 1955, 8 (11), 710−715.(13) Kaplan, D. L.; Mello, C. M.; Arcidiacono, S.; Fossey, S.; Senecal,K.; Muller, W. Silk. In Protein-Based Materials; McGrath, K., Kaplan,D., Eds.; Birkhauser: Boston, 1997.(14) Miller, L. D.; Putthanarat, S.; Eby, R. K.; Adams, W. W.Investigation of the nanofibrillar morphology in silk fibers by smallangle X-ray scattering and atomic force microscopy. Int. J. Biol.Macromol. 1999, 24 (2−3), 159−165.(15) Putthanarat, S.; Stribeck, N.; Fossey, S. A.; Eby, R. K.; Adams,W. W. Investigation of the nanofibrils of silk fibers. Polymer 2000, 41(21), 7735−7747.(16) Jin, H. J.; Kaplan, D. L. Mechanism of silk processing in insectsand spiders. Nature 2003, 424 (6952), 1057−1061.(17) Altman, G. H.; Diaz, F.; Jakuba, C.; Calabro, T.; Horan, R. L.;Chen, J.; Lu, H.; Richmond, J.; Kaplan, D. L. Silk-based biomaterials.Biomaterials 2003, 24 (3), 401−416.(18) Sen, K.; Babu, K. M. Studies on Indian silk. I. Macro-characterization and analysis of amino acid composition. J. Appl. Polym.Sci. 2004, 92 (2), 1080−1097.(19) Martel, A.; Burghammer, M.; Davies, R. J.; Di Cola, E.;Vendrely, C.; Riekel, C. Silk fiber assembly studied by synchrotronradiation saxs/waxs and Raman spectroscopy. J. Am. Chem. Soc. 2008,130 (50), 17070−17074.(20) Hu, X.; Kaplan, D.; Cebe, P. Dynamic protein-waterrelationships during beta-sheet formation. Macromolecules 2008, 41(11), 3939−3948.(21) Rodin, V. V.; Knight, D. P. The molecular mobility of water innatural polymers: Silk Bombyx mori with a low water content asstudied by H-1 DQF NMR. Biofizika 2004, 49 (5), 800−808.(22) Hamedi, M.; Forchheimer, R.; Inganas, O. Towards woven logicfrom organic electronic fibres. Nat. Mater. 2007, 6 (5), 357−362.(23) Aurora Silk, 434 Northeast Buffalo Street, Portland, OR.(24) Kraft, P.; Bergamaschi, A.; Bronnimann, C.; Dinapoli, R.;Eikenberry, E. F.; Graafsma, H.; Henrich, B.; Johnson, I.; Kobas, M.;Mozzanica, A.; Schleputz, C. A.; Schmitt, B. Characterization andcalibration of Pilatus detectors. IEEE Trans. Nucl. Sci. 2009, 56 (3),758−764.(25) Guizar-Sicairos, M.; Diaz, A.; Holler, M.; Lucas, M. S.; Menzel,A.; Wepf, R. A.; Bunk, O. Phase tomography from x-ray coherentdiffractive imaging projections. Opt. Express 2011, 19 (22), 21345−21357.(26) Nagano, A.; Kikuchi, Y.; Sato, H.; Nakazawa, Y.; Asakura, T.Structural characterization of silk-based water-soluble peptides (Glu)-(n)(Ala-Gly-Ser-Gly-Ala-Gly)(4) (n = 4−8) as a mimic of Bombyxmori silk fibroin by C-13 solid-state NMR. Macromolecules 2009, 42(22), 8950−8958.(27) Wand, M. P.; Jones, M. C. Kernel Smoothing; Chapman & Hall/CRC: Boca Raton, FL, 1995.(28) Squires, T. M.; Quake, S. R. Microfluidics: Fluid physics at thenanoliter scale. Rev. Mod. Phys. 2005, 77 (3), 977−1026.(29) Holler, M.; Raabe, J.; Diaz, A.; Guizar-Sicairos, M.; Quitmann,C.; Menzel, A.; Bunk, O. An instrument for 3D X-ray nano-imaging.Rev. Sci. Instrum. 2012, 83 (7), 073703−7.(30) Thibault, P.; Guizar-Sicairos, M. Maximum-likelihood refine-ment for coherent diffractive imaging. New J. Phys. 2012, 14 (6),063004.(31) Schropp, A.; Hoppe, R.; Patommel, J.; Samberg, D.; Seiboth, F.;Stephan, S.; Wellenreuther, G.; Falkenberg, G.; Schroer, C. G. Hard X-ray scanning microscopy with coherent radiation: Beyond the

Macromolecules Article

dx.doi.org/10.1021/ma3021163 | Macromolecules 2013, 46, 434−439438

resolution of conventional X-ray microscopes. Appl. Phys. Lett. 2012,100 (25), 253112−3.(32) Vila-Comamala, J.; Diaz, A.; Guizar-Sicairos, M.; Mantion, A.;Kewish, C. M.; Menzel, A.; Bunk, O.; David, C. Characterization ofhigh-resolution diffractive X-ray optics by ptychographic coherentdiffractive imaging. Opt. Express 2011, 19 (22), 21333−21344.

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