Journal of Structural Biology 184 (2013) 454–463

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Journal of Structural Biology

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Crystal nucleation and near-epitaxial growth in nacre

1047-8477/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.jsb.2013.10.002

⇑ Corresponding author at: Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706, USA.

E-mail address: pupa@physics.wisc.edu (P.U.P.A. Gilbert).

Ian C. Olson a, Adam Z. Blonsky a, Nobumichi Tamura b, Martin Kunz b, Boaz Pokroy c, Carl P. Romao d,Mary Anne White d, Pupa U.P.A. Gilbert a,e,⇑a Department of Physics, University of Wisconsin-Madison, 1150 University Avenue, Madison, WI 53706, USAb Advanced Light Source, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USAc Department of Materials Science & Engineering, and The Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa 32000, Israeld Department of Chemistry and Institute for Research in Materials, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canadae Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 2 July 2013Received in revised form 3 October 2013Accepted 5 October 2013Available online 10 October 2013

Keywords:BiomineralMolluscaTabletAragoniteBridge tiltingEpitaxyEpitaxialLow-angle grain boundaryMesocrystalPIC-mappingXANESPEEMMicro-X-rayDiffraction

Nacre is the iridescent inner lining of many mollusk shells, with a unique lamellar structure at the sub-micron scale, and remarkable resistance to fracture. Despite extensive studies, nacre formation mecha-nisms remain incompletely understood. Here we present 20-nm, 2�-resolution polarization-dependentimaging contrast (PIC) images of shells from 15 mollusk species, mapping nacre tablets and their orien-tation patterns. These data show where new crystal orientations appear and how similar orientationspropagate as nacre grows. In all shells we found stacks of co-oriented aragonite (CaCO3) tablets arrangedinto vertical columns or staggered diagonally. Near the nacre-prismatic (NP) boundary highly disorderedspherulitic aragonite is nucleated. Overgrowing nacre tablet crystals are most frequently co-orientedwith the underlying aragonite spherulites, or with another tablet. Away from the NP-boundary all tabletsare nearly co-oriented in all species, with crystal lattice tilting, abrupt or gradual, always observed andalways small (plus or minus 10�). Therefore aragonite crystal growth in nacre is near-epitaxial. Basedon these data, we propose that there is one mineral bridge per tablet, and that ‘‘bridge tilting’’ may occurwithout fracturing the bridge, hence providing the seed from which the next tablet grows near-epitaxially.

� 2013 Elsevier Inc. All rights reserved.

1. Introduction

The complex arrangement of lamellar tablets and organic sheetsin nacre inspires biomimetic materials (Amini and Miserez, 2013;Bonderer et al., 2008; Meyers et al., 2013; Munch et al., 2008; Ortizand Boyce, 2008) yet its formation mechanisms are poorly under-stood (Addadi et al., 2006; Belcher et al., 1996; Mutvei, 1979; Pok-roy et al., 2007a,b; Schäffer et al., 1997; Wada, 1972; Weiner andHood, 1975; Wise, 1970). It is well established that interlamellarorganic sheets of b-chitin and proteins (Addadi and Weiner,1997; Belcher et al., 1996; Mutvei, 1979) are deposited first (Beve-lander and Nakahara, 1969; Levi-Kalisman et al., 2001), then spaceis filled by growing aragonite (CaCO3) tablets (Bevelander andNakahara, 1969; Nakahara, 1979). However, many different

models have been proposed for the mechanisms of nacre forma-tion, as summarized in Fig. 1. In this and all other figures in thiswork, nacre is always growing upward, hence under- or over-lyingnacre tablets mean previously- or subsequently-formed,respectively.

Weiner et al. proposed that organic sheets template aragonitetablet orientation by heteroepitaxy (Weiner and Hood, 1975; Wei-ner and Lowenstam, 1986; Weiner and Traub, 1980; Weiner et al.,1984). The idea that organic molecules enact polymorph selectionand crystal orientation, collectively referred to here as ‘‘Weinertemplates’’, was validated, at least for the polymorph selectionpart, when organic molecules were shown to induce aragoniterather than calcite growth in vitro (Belcher et al., 1996; Faliniet al., 1996; Metzler et al., 2010). Another hypothesis by Nudelmanet al. had each tablet crystal nucleated independently, by a single,well-defined protein arrangement under each tablet termed‘‘nucleation site’’ (Nudelman et al., 2006). ‘‘Nudelman sites’’ arehighly conserved across species (Nudelman et al., 2006). No

Fig. 1. Schematics of models proposed for nacre formation mechanisms. (A) In the ‘‘Weiner template’’ model an organic layer of ordered molecules (magenta) imparts theorientation to the overgrowing aragonite crystal tablet (blue) (Weiner and Hood, 1975; Weiner and Lowenstam, 1986; Weiner and Traub, 1980; Weiner et al., 1984). Thediagonal black dashing represents the orientation, which is templated heteroepitaxially. (B) An organic ‘‘Nudelman site’’ (magenta box) approximately 1-lm in diameterinitiates the growth of one aragonite tablet (blue box) (Nudelman et al., 2006). (C) A myriad of ‘‘Schäffer bridges’’ homoepitaxially connect two adjacent tablets (blue) byextending through pores in the organic sheet (magenta) separating two tablets (Schäffer et al., 1997). The ‘‘Schäffer bridges’’ were ruled out by Checa et al. (2011). (D) Metzleret al. (2007) hypothesized that stop/start molecule (red and green, respectively) are in the immediate vicinity of one another and they work in tandem. They are on either sideof the same organic sheet, with stop molecule in underlying layer N and start molecule in layer N + 1. When growing aragonite crystal N comes in contact with the stopmolecule its growth is arrested, and the start molecule initiates the growth of aragonite crystal N + 1, co-oriented with crystal N. (E) ‘‘Checa bridges’’ (blue) were found to be�200 nm in diameter, extend through holes in the organic sheet (magenta) between two subsequent tablets (blue), which are co-oriented (black dashing) (Checa et al., 2011).(F) The new model proposed by Gilbert, in which one ‘‘Checa bridge’’ extends through one organic ‘‘Nudelman site’’ per nacre tablet (Olson et al., 2012). Organic sheets arerepresented by magenta lines, ‘‘Nudelman sites’’ by thicker magenta donuts with a central hole, and tablets are blue. Black dashing indicates that all three tablets are co-oriented crystals, each growing epitaxially from the underlying one. (For interpretation of the references to color in this figure legend, the reader is referred to the web versionof this article.)

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statements were made about crystal orientations. Two strong anduseful statements were introduced by Nudelman et al.: there is asingle ‘‘Nudelman site’’ per tablet, and the ‘‘Nudelman site’’ isorganic.

A third hypothesis by Schäffer et al. had all tablets extendinginto a myriad of ‘‘mineral bridges’’ through pores in the organicsheets (�100 pores/lm2, thus an equal density of ‘‘Schäfferbridges’’ was inferred), an extension of the connected tablet modeldescribed by Wada (1972), thus crystal growth would be homo-epitaxial, from the underlying tablet (Schäffer et al., 1997). Re-cently Checa et al. showed that the small ‘‘Schäffer bridges’’ innacre from various mollusk species do not connect tablets—theyare interrupted by organics, and the crystals across interruptionsare not co-oriented (Checa et al., 2011). They also showed larger�200 nm wide mineral bridges near the center of tablets in gastro-pods and cephalopods, or near a tablet edge in bivalve nacre (Checaet al., 2011). The number of ‘‘Checa bridges’’ that exist in each tab-let could not be addressed by that study. Independent of Checa,Gilbert proposed a mechanism for nacre growth in which a single‘‘Checa bridge’’ forms at the center of each organic ‘‘Nudelmansite’’, and therefore represented the ‘‘Nudelman sites’’ as donuts,with a hole at their center (Olson et al., 2012) (Fig. 1F). Metzleret al. (2007) hypothesized a different mechanism, with organicstop/start molecules that work in tandem, with the stop and thestart molecules in layers N and N + 1, respectively. When growingaragonite crystal N comes in contact with the stop molecule its

growth is arrested, and the start molecule starts the growth of ara-gonite crystal N + 1, co-oriented with crystal N (Fig. 1D). The stop/start organic molecules have not been identified. In Fig. 1A and Bmodels it is unclear how the time sequence of nacre growth couldbe established. All other models (C–F in Fig. 1) enforce the time se-quence, which is key to prevent shell voids and mechanical failure.Specifically in Fig. 1D, no start molecule can nucleate tablet crys-tals N + 1, unless the underlying stop molecule has been reachedby crystal N. Similarly, in Fig. 1E and F, tablet N + 1 cannot growfrom a ‘‘Checa bridge’’ before tablet N.

Previous work left key questions unanswered: What is themechanism of aragonite nucleation in each nacre tablet? Doesnucleation exhibit any differences with location in the shell?How is the crystal orientation of a tablet transmitted to the overly-ing one? The data here provide direct evidence and suggest new in-sights into these fundamental aspects of nacre crystal nucleation,orientation and growth, in a suite of mollusk species.

2. Brief methods

2.1. Samples

The shells of fifteen mollusk species were cut, embedded inepoxy (either EpoThin, Buehler, IL, or EpoFix, Electron MicroscopySciences, PA), polished perpendicularly to the nacre layers withdecreasing size alumina grit down to 50 nm (MasterPrep, Buehler,

456 I.C. Olson et al. / Journal of Structural Biology 184 (2013) 454–463

IL), and coated with Pt using a sputter coater (208HR, Cressington,UK). Samples were analyzed with PEEM-3 on beamline 11.0.1 andLaue micro-X-ray diffraction on beamline 12.3.2 at the AdvancedLight Source at Lawrence Berkeley National Laboratory in Berkeley,CA. Detailed methods are provided in the Supporting informationonline.

2.2. PIC-mapping with PEEM

Photoelectron emission spectromicroscopy (PEEM) (De Stasioet al., 1992a,b, 1993a,b; Gilbert et al., 2003; Gong et al., 2012)was used to collect PIC-maps as described in Gilbert et al. (2011),Killian et al. (2009, 2011), Metzler et al. (2007, 2008a, 2010) andreviewed in Gilbert (2012, 2014). Nineteen images were collectedat the same 290.3 eV photon energy, and a sample voltage of�15 kV, while the linear X-ray polarization vector was rotated be-tween 0� and 90� in 5� increments. Each pixel of these stacks of 19images, therefore, contained a polarization-dependence curvewhich was fit to the function y = a + b cos(EPU� + c). The analysiswas repeated for all 106 pixels in each stack of 20 lm � 20 lmimages, with 20-nm pixel size, producing a PIC-map image inwhich the fit parameters c were displayed as quantitative gray lev-els. Angle spread measurements were taken as the ‘‘footprint’’ ofthe distribution of angles in a PIC-map.

2.3. lXRD analysis

Samples were illuminated with ‘‘pink beam’’ X-rays of photonenergies ranging between 5 < hm < 22 keV. X-ray microdiffractionpatterns were obtained using a Pilatus 1 M X-ray detector. LaueX-ray microdiffraction patterns were indexed using the XMAS soft-ware (Tamura et al., 2003). Indexing provides the full 3-dimen-sional orientation matrix for each crystal, as well as distributionmaps of aragonite crystallite orientations in the sample (Tamuraand Gilbert, 2013).

2.4. Finite element analysis

Tilted aragonite bridges were modeled using COMSOL Multi-physics 4.2, at Dalhousie University, Halifax, Nova Scotia, Canada.

3. Results and discussion

Using polarization-dependent imaging contrast (PIC) mapping(Gilbert, 2012) at the nano-scale (Gilbert et al., 2011; Olson et al.,2012), we analyzed shells from 15 species with representative re-sults displayed in Fig. 2 and Supporting information Fig. S1. The 15species include: the marine bivalves Atrina rigida (Ar), Mytilus cal-ifornianus (Mc), Mytilus edulis (Me), Mytilus galloprovincialis (Mg),Pinctada fucata (Pf), Pinctada margaritifera (Pm), the marine gastro-pods Haliotis discus (Hd), Haliotis iris (Hi), Haliotis laevigata (HL),Haliotis pulcherrima (Hp), Haliotis rubra (Hrb), Haliotis rufescens(Hrf), the marine cephalopod Nautilus pompilius (Np), and the fresh-water bivalves Lasmigona complanata (Lc), and Pyganodon grandis(Pg). All marine bivalves and gastropods considered in this studyhave a calcite prismatic layer, while the freshwater bivalves andmarine cephalopod have an aragonite prismatic layer.

In a PIC-map the gray level corresponds to aragonite or calcite c-axis orientation, and the patterns of orientations of micro-crystal-line tablets provide insights into the formation of nacre. Fig. 2shows with unprecedented detail the transition from the calciteprismatic layer to the aragonite nacre layer in Pf, Hi, and Np. Inthese shells, spherulitic aragonite is revealed by PIC-mapping be-tween the prismatic layer and the first layer of lamellar nacre(No). Notice that the orientation of tablets at No is identical to

the orientation of the underlying spherulites, as previously ob-served in Np (Mutvei, 1972), Hr (Gilbert et al., 2008; Su et al.,2002), and Pf (Metzler et al., 2010). It is evident from the imagesin Fig. 2 that nacre tablet orientation contrast in Pf, Hi, and Np de-cays with distance from No. Images of the nacre-prismatic (NP)-boundary in 12 additional species are presented in Supportinginformation Figs. S1 and S2.

Until recently, nacre in all species was assumed to have co-ori-ented aragonite crystal c-axes, perpendicular to the shell surface(Suzuki et al., 2009). Many authors observed mis-orientations innacre c-axes, e.g. 10� (DiMasi and Sarikaya, 2004), but attributedthem to the macroscopic shell curvature. Other authors showeddisorder in nacre near the NP-boundary (Checa and Rodriguez-Navarro, 2001; Fritz et al., 1994; Metzler et al., 2008b), that disor-der decreases away from the boundary (Gilbert et al., 2008; Schnei-der et al., 2012), and that neighboring stacks of nacre tablets couldhave significantly different orientation (Dalbeck et al., 2006; Met-zler et al., 2007). The new data in Fig. 2 are consistent with all theseprevious observations.

Striking differences between species in Fig. 2 and Supportinginformation Figs. S1 and S2, are revealed by PIC-mapping: the mor-phology of the prismatic layer, the presence or absence of spheru-litic aragonite, the arrangement of co-oriented nacre tablets, thedegree of crystal mis-orientation, and gradual changes in nacrecrystalline order as a function of distance from No (Gilbert et al.,2008). Columnar nacre (Addadi and Weiner, 1997; Cartwrightet al., 2009; Metzler et al., 2007) from the gastropod (Hd, Hi, HL,Hp, Hrb, Hrf) and cephalopod (Np) shells in PIC-maps showsstraight vertical columns of co-oriented tablets. Sheet nacre (Cart-wright et al., 2009; Rousseau et al., 2005) in the bivalve shells (Ar,Lc, Mc, Me, Mg, Pf, Pg, Pm) shows stacks of a few co-oriented tabletsstaggered diagonally, most clearly visible in Pf (Fig. 2B).

Gilbert et al. showed that, in Hrf nacre, aragonite tablet c-axesgradually order along the nacre growth direction as distance fromthe nacre-prismatic (NP)-boundary increases (Gilbert et al., 2008).This qualitative observation was explained with a model in whichfaster-growing tablets have their c-axes along the growth direc-tion, and gradually prevail in a competition for space. Here wequantitatively measure the angle spread in each series of PIC-maps,defined as the footprint of the histogram of all c0-axis angles (Olsonet al., 2012), and plot angle-spreads as a function of distance fromNo for all species as shown in Fig. 3. The c0-axis is defined as theprojection of the c-axis onto the 2D plane perpendicular to theX-ray propagation direction, that is, the plane in which the electricfield vector oscillates. This is described with a schematic in Ref.(Olson et al., 2012). The c0-axis not the c-axis orientation is mea-sured by PIC-mapping. Specifically, the angle between the c’-axisand the vertical of the laboratory is quantitatively represented bydifferent graylevels, according to the gray scale in each PIC-map.The footprint of the histogram of all c0-axes in a 20 lm � 20 lm re-gion of nacre is the measured angle spread (e.g. 33� in Supportinginformation Fig. S9A). The 13 shell species in which decay of anglespread is observed have convergence distances 20–400 lm, hencethis behavior is typical, and the previously reported 50 lm for Hrf(Gilbert et al., 2008) is reproduced here (Supporting informationTable S1).

3.1. Aragonite crystal nucleation near the NP-boundary

Fig. 3 depicts the angular spread of nacre as a function of dis-tance from No. It provides evidence for the nucleation of disor-dered, randomly oriented crystals near the NP-boundary in 8species (c0-axes spread by 180�, Fig. 3A), more ordered crystals(60�, Fig. 3B) in 5 others species, whereas in 2 species the crystalshave c0-axes ordered within 30�, which is as ordered as nacre dis-tant from the NP-boundary (30�, Fig. 3C).

Fig. 2. Multi-resolution images of shell cross-sections at the nacre-prismatic (NP)-boundary in the bivalve Pinctada fucata (Pf), the gastropod Haliotis iris (Hi) and thecephalopod Nautilus pompilius (Np). (A, C, E) Polarized, reflected light micrographs illustrating (bottom) the prismatic calcite (in Pf, Hi) or aragonite (in Np) layer, and (top) thearagonite nacre layer. (B, D, F) PIC-maps of the NP-boundary and nacre in the three species, with 20-nm pixels and 2� angle resolution. The grayscale bars at the bottomindicate the angle between the aragonite c0-axis of each crystal and the nacre growth direction (vertical, from bottom to top in all panels). The angles ranging from�90� to 90�are displayed in black and white, respectively, 0�, that is, vertical c0-axis in the plane of the figure, which is also the vertical of the laboratory during data acquisition, is gray.The gray level contrast in both types of images is due to different calcite or aragonite c-axis orientations. Contrast decreases with distance from the first nacre layer in thesespecies. Not coincidentally, in all three shells there is disordered spherulitic aragonite at the NP-boundary. All scale bars are 10 lm.

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The angle spread of c0-axes in nacre, away from the NP-bound-ary, is �30� for all 15 species analyzed here (Fig. 3 and Supportinginformation Table S1).

These data show that nucleation of disordered spherulitic ara-gonite crystals, or aragonite prismatic layer crystals in Np, Lc, andPg, occurs near the NP-boundary. In bulk nacre, away from theboundary, however, highly disordered tablet orientations are notobserved. Tablet nucleations in bulk nacre, therefore, must havecontrolled crystal orientations, and be different from those nearthe boundary, which exhibit orientational disorder (180�, 60� c0-axis spread) in 13 of the 15 species analyzed.

The formation of ordered nacre at No in the other 2 species, Pgand Ar, has different interpretations. Since the prismatic layer in Pgis aragonitic, and the prisms exhibit c0-axis angle spread within30�, the orientation of tablets in the initial nacre layers is consis-tent with near-epitaxial growth over the ordered prisms (Pokroyand Zolotoyabko, 2005). This is also consistent with the observa-tions of Freer et al. in another freshwater mussel (Freer et al.,2009). In Ar instead the prismatic layer is calcitic, thus Ar mustcontrol crystal orientation at the onset of nacre deposition, possi-bly by a ‘‘Weiner template’’ mechanism (Weiner et al., 1984) orby inhibiting the growth of crystals with undesirable orientations(Addadi and Weiner, 1985). This is also the case, although to a les-ser extent, in 5 other species (Pf, Lc, Np, Pm, Mc), in which the crys-tal orientation angle spread at No is not random but �60� and

decays to �30� (Supporting information Table S1). A non-zeroamount of crystal orientation control, therefore, must be exertedupon nucleation near the NP-boundary. This is consistent withthe crystal orientation control found by Suzuki et al. for the Pif pro-tein in Pf (Suzuki et al., 2009). For the remaining 8 species (Mg, Hp,HL, Hi, Hrf, Hd, Me, Hrb), c0-axis angle spread shows that orienta-tions are initially random (�180�). Micro-diffraction results on 3shells confirm these observations. The spread of c-axes at and awayfrom the NP-boundary is 180–20� in Hrf, 78–20� in Pm, and in Arthe c-axis angle spread is �20� and constant (Supporting informa-tion Fig. S3). The minor discrepancies between measured c-axisand c0-axis angle spread values likely result from sampling differ-ences or geometry, as explained in Supporting informationFigs. S3 and S4. The c-axis angle spreads observed here are inagreement with previous X-ray diffraction data obtained for redabalone by measuring the footprint of a rocking curve (22�) (Met-zler et al., 2008a), or the footprint of a pole figure (32�) (Checaet al., 2005; Zaremba et al., 1996), or the footprint (22�) of a rockingcurve in another seawater mussel (Younis et al., 2012).

3.2. Epitaxial crystal growth near the NP-boundary

Fig. 4 presents high-resolution PIC-maps of the NP-boundaries,which exhibit significant differences across the 15 mollusk species.In many of the species in Fig. 4, tablets at No have the same

Fig. 3. Crystals are nucleated near the NP-boundary, and are most disordered there,then they gradually order in most species. Measurements of angle spread as afunction of distance from the first layer of lamellar nacre (No) in 18 shells from 15species are presented here. Angle spread measurements were obtained from20 lm � 20 lm PIC-maps of nacre. In a PIC-map, gray level indicates c0-axisorientation. A histogram of all gray levels, therefore, provides the distribution of allangles in a PIC-mapped region of nacre. The angle spread is the full width at thebase of this distribution. The data for each species were fit to an exponential decay(A and B) or to a constant (C). Species names are abbreviated as indicated in the textfootnote. All fit parameters are in Supporting information Table S1. Away from No

all 15 species have similar angle spreads: 30� ± 10� (mean ± Std. Dev.). Near No theshells differ: most disordered (180� red, A), intermediate (60� green, B), ordered(30� blue, C). (For interpretation of the references to color in this figure legend, thereader is referred to the web version of this article.)

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orientation as the underlying aragonite. This is most strikingly evi-dent in the PIC-maps from all Haliotis (Hp, HL, Hi, Hrf, Hd, Hrb) andPinctada (Pf, Pm), but also in some of the Mytilus (Mg, Me but notMc), Lc, Np, and Pg shells. In 13 of the 15 species the angle-spreaddecay behavior observed in Fig. 3 is correlated with the existenceof disordered spherulitic aragonite at the NP-boundary (Fig. 4and Supporting information Table S1). Although mis-orientationssmaller than 2� would not be appreciated in PIC-maps, the obser-vation of c0-axis orientation shared between nacre tablets and theunderlying aragonite spherulites (Figs. 2 and 4, Supporting infor-mation Figs. S1 and S2) suggests that aragonite crystals growhomoepitaxially across nacre organic sheets. Further support for

homoepitaxy was found in an unusual location of the Np shell,where striking transitions occur from nacre to spherulitic arago-nite, to nacre again, in which the crystal orientations are consis-tently homoepitaxial across nacre interlamellar organic sheets, asshown in Supporting information Fig. S6. Pores in the organicsheets (Fig. 1F) provide a simple explanation for the observed crys-tal growth of aragonite, which is nearly-co-oriented, within 2�.Mis-orientations smaller than 2� would not be appreciated inPIC-maps. More complex mechanisms such as that described byMetzler et al. (2007) (Fig. 1D) are also plausible, but do not explainthe ‘‘Checa bridges’’ so clearly and reproducibly shown by Checaet al. (2011), and also by Gries et al. (2009). We recently proposedthat ‘‘Checa bridges’’ through such holes topologically connect tab-lets in a stack (Olson et al., 2012). The data presented here supportthat hypothesis: most frequently a stack of tablets is co-oriented,with c-axis orientation alignment of 2� or better, in agreementwith TEM observations (Checa et al., 2011; Gries et al., 2009; Youn-is et al., 2012). The same extent of alignment is expected for the a-and b-axes.

3.3. Near-epitaxial crystal growth away from the NP-boundary

High resolution PIC-maps of nacre away from the NP-boundaryshow crystal lattice tilting in Figs. 2 and 5, Supporting informationFigs. S1, S7, and S8. The tilting of crystal lattice away from theboundary is evident by changes abrupt or gradual in gray levelsin all the PIC-maps. These are not homogenous in orientation awayfrom the NP-boundary, thus they reveal that crystal growth in bulknacre is more complex than the simple homoepitaxy observed nearthe NP-boundary.

The changes in tablet orientation shown in Figs. 2 and 5, Sup-porting information Figs. S1, S2, S7, and S8 differ across classesof nacre-forming mollusks: in bivalve sheet nacre there are onlyabrupt or no gray level changes from tablet to tablet, whereas ingastropod and cephalopod columnar nacre the changes can be alsogradual, as shown by gradients of color or gray level in PIC-mapswithin the same column of tablets. Columnar nacre, therefore,shows either no change, abrupt, or gradual change in table orienta-tion within each nacre column. Although crystal lattice tilting waspreviously observed in prismatic calcite (Checa et al., 2013; Olsonet al., 2013) and prismatic aragonite (Pokroy and Zolotoyabko,2003), and in dendritic growth of synthetic systems (Ming et al.,1993; Wang et al., 1998, 2001), this is the first evidence of crystallattice tilting in nacre aragonite crystals, and is extensively andconsistently observed in all PIC-maps, as presented in Figs. 2 and5, Supporting information Figs. S1, S2, S7, and S8.

Whether abrupt or gradual, the orientation changes observed inbulk nacre, and termed crystal lattice tilting here, are always small,with c-axis spread within ±10�, consistent across all PIC-maps, allspecies, and in agreement with diffraction data (Supporting infor-mation Figs. S3).

3.4. ‘‘Bridge tilting’’ hypothesis

Since the crystal lattice tilting observed is always limited, onecan think of a growing stack of tablets as a topologically connectedsingle crystal, with only small (±10�, Supporting informationFig. S3) crystal lattice tilting even when comparing tablets thatare millimeters apart in the same stack. Such a stack of tabletswould contain low-angle grain boundaries (Johnson et al., 2004).Stacks of nearly-co-oriented tablets are staggered diagonally insheet nacre and stacked vertically in columnar nacre. Whatconnects two immediately subsequent tablets in a stack? The pres-ent PIC-mapping data do not directly observe a topological connec-tion. Our hypothesis to interpret the data is that a single ‘‘Checabridge’’ per tablet, slightly tilted in orientation as it grows, seeds

Fig. 4. PIC-maps at the NP-boundary in 15 mollusk species show that nacre grows epitaxially from underlying spherulitic aragonite. The c0-axis orientations, displayed as graylevels, show dramatic differences across species. All panels display the prismatic layer at the bottom and nacre at the top. The prismatic layer is calcite in all species, except forPg, Lc, Np, and Hp, where it is entirely aragonite. In Hd it is mostly calcite, with additional spherulitic aragonite at the outermost surface of the shell (Supporting informationFig. S5). Species names are abbreviated as indicated in the text. The shell name colors (red, green, blue) are consistent with those defined in Fig. 3, and also used in all other PICmaps here and in the Supporting information Figs. S1, S2, Table S1. All images share the same 5-lm scale bar, and the same gray level bar shown at the bottom. (Forinterpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5. False-colored PIC-map of nacre in Haliotis laevigata. Stacks of co-orientedtablets are homogeneously colored, whereas the central dark stack exhibits agradient of colors. Additional data showing abrupt or gradual changes in crystalorientations in the same stack or column of nacre tablets are presented inSupporting information Figs. S7 and S8.

Fig. 7. (A) Model of one monolithic ‘‘Checa bridge’’ under shear stress, with all theresultant strain in the bridge. (B) Strain energy of a ‘‘Checa bridge’’ with its c-axistilted towards the a- or b-axis, compared to the additional surface energy of twonew faces that would result from fracture of the bridge, that is, the energy requiredfor fracture to occur. The details of this simulation are described in Section 2, in theSupporting information, and Fig. S10.

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the orientation of the next tablet. Tilting in the same or a differentdirection may be repeated at the next bridge and tablet. Hence at‘‘Checa bridges’’ there would often, perhaps always be low-anglegrain boundaries (Johnson et al., 2004; Kobayashi et al., 2005).Nucleation events similar to those observed near the NP-boundarywould have c0-axis angle spreads in the range between ±30� and±90� (Fig. 3) and is therefore ruled out. A new and different nucle-ation event, with imperfectly aligned ‘‘Weiner templates’’ in ‘‘Nud-elman sites’’ is consistent with the observed data, but in this casethe molecules and mechanisms responsible for templating arago-nite orientation remain to be discovered. Organic bridges such asthe stop/start hypothetical molecules described by Metzler et al.(2007) (Fig. 1D) could also explain the observed small changes,

Fig. 6. Model for nacre near-epitaxial crystal growth via ‘‘bridge tilting’’. Schematic reprgrowth. The bottom tablet formed first and is broader, the others have decreasing widthepitaxially through ‘‘Checa bridges’’, as shown in Fig. 1E. The black dashing is verticaconnecting two tablets. Hence the subsequent patterns of lines are 2�, 4�, 6�, . . .. , 20� tilteGrowth via ‘‘bridge tilting’’ results in crystal lattice tilting, and is termed ‘‘near-epitaxia

but to date there is no evidence for the existence of these hypo-thetical molecules. Because hard evidence for ‘‘Checa bridges’’ ex-ists (Checa et al., 2011), the most plausible explanation is ‘‘bridgetilting’’ as described in detail in Fig. 6.

In cases such as the dark column in Fig. 5, stacks of tablets showa gradient of gray levels, rather than the more common co-orientedstacks. A possible explanation for this phenomenon assumes a sin-gle ‘‘Checa bridge’’, which is relatively small (�200 nm). When the‘‘Checa bridge’’ protrudes as the one at the top of the schematic ofFig. 6, it is exposed, fragile, and can easily be slightly pushed orpulled by the mantle in the living and moving mollusk. Eventhough the protruding ‘‘Checa bridge’’ is covered by an organicmembrane (Nakahara, 1979), this is likely to be mechanically

esentation of ‘‘bridge tilting’’ in one stack of 11 tablets in columnar nacre during itss, will grow horizontally until they abut other tablets, and vertically they will growl in the bottom tablet, and then tilts in orientation by 2� at each ‘‘Checa bridge’’d with respect to the bottom pattern, whereas the tablet surfaces remain horizontal.l’’ in this work.

I.C. Olson et al. / Journal of Structural Biology 184 (2013) 454–463 461

compliant, thus when exposed, the free-standing ‘‘Checa bridge’’ atthe top is in mechanical contact with the mantle cells of the mol-lusk, and may be vulnerable to mantle contraction in a specificdirection, resulting in bridge tilting or breaking. The subsequenttablet will grow co-oriented with its starting crystal seed, that is,the top of a ‘‘Checa bridge’’, in a tilted or un-tilted orientation.Thus, if there is a systematic push or pull by the mantle in onedirection, subsequent tablets will still have their top and bottomsurfaces parallel to all others and the usual layered nacre appear-ance will be observed, but the crystal lattice orientations of subse-quent tablets will be gradually changing, resulting in theorientation gradient observed in Fig. 5 and schematically repre-sented in Fig. 6. This crystal lattice tilting mechanism is best ex-plained with a single ‘‘Checa bridge’’ per tablet.

We stress that the ‘‘bridge tilting’’ in Fig. 6 is, at this time, only ahypothesis. We do not have direct evidence for bridge tilting. Fu-ture high-resolution transmission electron microscopy (Checaet al., 2011) or electron back scattered diffraction (EBSD) (Checaet al., 2009; Dalbeck et al., 2006; MacDonald et al., 2010; Perez-Huerta et al., 2011; Schmahl et al., 2012) studies of nacre couldprovide direct evidence to test the hypothesis of ‘‘bridge tilting’’.

We assessed the plausibility of a ‘‘bridge tilting’’ mechanisminvolving mechanical force on the bridges by finite element analy-sis, as shown in Fig. 7. We found that tilting the c-axis of an arago-nite cylinder the size of a ‘‘Checa bridge’’ by up to approximately 4�is unlikely to cause the bridge to fracture. This is a conservativeestimate, as the energy required to fracture a crystal is at leastan order of magnitude greater than the surface energy of two crys-tal faces resulting from fracture (Anderson, 2005). Bridge tilting by±10�, therefore, is a realistic hypothesis for both the abrupt and thegradual changes of crystal orientations observed in all speciesaway from the NP-boundary.

The observed crystal lattice tilting is consistent either withnacre crystals forming from ions in solution, or from a proteingel (Keene et al., 2010), or from amorphous precursor phases (Add-adi et al., 2006; Nassif et al., 2005). In any of these scenarios thebridge tilting hypothesis is equally realistic.

3.5. One ‘‘Checa bridge’’ per tablet

How many ‘‘Checa bridges’’ are there per tablet? Independentlines of evidence suggest that there is a single bridge per tablet:the Voronoi construction of tablets in each nacre layer (Gilbertet al., 2008; Rousseau et al., 2005) can only be formed if there isa single seed for the growth of each tablet; If there is one ‘‘Nudel-man site’’ and one ‘‘Checa bridge’’ per tablet, these must be thesame entity. A single ‘‘Checa bridge’’ per tablet is consistent withthe crystal lattice tilting observed here, and its possible explana-tion by ‘‘bridge tilting’’, in Fig. 6. We stress that one ‘‘Checa bridge’’per tablet is a logical deduction, not an experimental observation,as the PIC-maps presented here did not directly reveal either ‘‘Nud-elman sites’’ or ‘‘Checa bridges’’ or ‘‘bridge tilting’’. This deduction,however, has the advantage of being based exclusively on real enti-ties, which have been directly observed (‘‘Nudelman sites’’, ‘‘Checabridges’’, strain in aragonite of the same size and extent of ‘‘bridgetilting’’), and not on hypothetical molecules that remain to be dis-covered. In this sense, the mineral ‘‘bridge tilting’’ mechanism pro-posed here is more realistic than an organic-mineral ‘‘Weinertemplate’’ (Fig. 1A) or organic–organic bridge (Fig. 1D). However,with the first complete mollusk genome recently published (Zhanget al., 2012), and complete proteomes of different mollusk shelllayers assembled (Marie et al., 2012), experimental determinationof functions of specific proteins or protein complexes during shellformation will be feasible in the near future, hence many doubtswill be resolved.

4. Conclusions

Clearly both nucleation and growth of crystals are highly regu-lated in mollusks. Because seawater, and presumably the extrapal-lial fluid from which nacre is formed (Crenshaw, 1972), is super-saturated with respect to aragonite, nucleation at random times,positions, and orientations must be actively inhibited. Sequential,connected, near-epitaxial aragonite crystal growth is enabled andregulated (Metzler et al., 2010; Suzuki et al., 2009) everywhere innacre, near and away from the NP-boundary, whereas nucleationof disordered aragonite crystal orientations is only observed nearthe boundary in 13 of the 15 species analyzed.

In all shells we found nearly co-oriented tablets stacked intocolumns or staggered diagonally, and in many cases these are co-oriented with underlying tablets or spherulitic aragonite within2�. Away from the NP-boundary, we observe abrupt or gradual,but invariably small (±10�) crystal lattice tilting. All tablets in astack, therefore, are nearly co-oriented, thus grow near-epitaxially.Based on these observations, we propose that one ‘‘Checa bridge’’,growing through one ‘‘Nudelman site’’, may be strained withoutfracturing, thus providing a seed for the growth and orientationof the next nacre tablet.

It is unclear at present whether greater co-orientation of nacretablets provides an evolutionary advantage to the mollusk. It istempting to conclude that such advantage exists, because theshells that do not start with ordered nacre rapidly achieve such or-der within the first 20–400 lm. But this may also be the result ofan abiotic mechanism, such as competition for space (Checaet al., 2006; Checa and Rodriguez-Navarro, 2005; Gilbert et al.,2008; Nys et al., 2004; Ubukata, 1994) and not provide anymechanical advantages to the mollusk. Perfect co-orientation,however, is best avoided. The crystal lattice tilting presented herefor the first time, and its possible formation mechanism via bridgetilting, would generate low-angle grain boundaries, which areknown to harden materials (Kobayashi et al., 2005). If confirmedexperimentally, such grain boundaries would confer a clearmechanical improvement to the shell and therefore an evolution-ary advantage to the mollusk.

Acknowledgments

We thank Sabine Gross for providing the Lc and Pg shells, andLisie Kitchel for the identification of these species. We thank SteveWeiner for the Hp shell, collected by Heinz Lowenstam, and iden-tified by David Lindberg. We thank ALS beamline scientists An-dreas Scholl and Anthony Young for their technical supportduring the PEEM-3 experiments, and Richard Celestre for samplepreparation. We thank Fabio Nudelman, Steve Weiner, and AmirBerman for reading the manuscript and suggesting improvements,and Lia Addadi for discussion. This work was supported by NSFAward DMR-1105167 and DOE Award DE-FG02-07ER15899 to PU-PAG and by NSERC grants to M.A.W. The experiments were per-formed at the Berkeley Advanced Light Source, supported by DOEunder contract DE-AC02-05CH11231.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jsb.2013.10.002.

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