NEUROSCIENCE. For the article ‘‘Rapid enhancement of two-stepwiring plasticity by estrogen and NMDA receptor activity,’’ byDeepak P. Srivastava, Kevin Woolfrey, Kelly A. Jones, Cassan-dra Y. Shum, L. Leanne Lash, Geoffrey T. Swanson, and PeterPenzes, which appeared in issue 38, September 23, 2008, of ProcNatl Acad Sci USA (105:14650–14655; first published September18, 2008; 10.1073�pnas.0801581105), the authors note that theauthor name Kevin Woolfrey should have appeared as Kevin M.Woolfrey. The author line has been corrected online. In addition,in the author contributions footnote and in the Acknowledg-ments, the initials K.W. should appear as K.M.W. The authorsalso note that due to a printer’s error, in Fig. 3A, some colorsprinted incorrectly. The corrected author line, and the correctedfigure and its legend, appear below.

Deepak P. Srivastava, Kevin M. Woolfrey, Kelly A. Jones,Cassandra Y. Shum, L. Leanne Lash, Geoffrey T. Swanson,and Peter Penzes

APPLIED BIOLOGICAL SCIENCES. For the article ‘‘Accurately quanti-fying low-abundant targets amid similar sequences by revealinghidden correlations in oligonucleotide microarray data,’’ byLuisa A. Marcelino, Vadim Backman, Andres Donaldson, Clau-dia Steadman, Janelle R. Thompson, Sarah Pacocha Preheim,Cynthia Lien, Eelin Lim, Daniele Veneziano, and Martin F.Polz, which appeared in issue 37, September 12, 2006, of ProcNatl Acad Sci USA (103:13629–13634; first published September1, 2006; 10.1073�pnas.0601476103), the authors note that in Eq.4, a �1 was inadvertently omitted from the denominator. Thedata in Fig. 1 were calculated using the correct equation, and thiserror in the published equation would make �1% difference inthe values of �. The corrected equation appears below.

�jk �1b �1 � 1�1 � ��1 � b��2 � 1� eh��Gjk�Gjj �1��, [4]

www.pnas.org�cgi�doi�10.1073�pnas.0809790105

BIOPHYSICS, CHEMISTRY. For the article ‘‘Transformation mecha-nism of amorphous calcium carbonate into calcite in the seaurchin larval spicule,’’ by Yael Politi, Rebecca A. Metzler, MikeAbrecht, Benjamin Gilbert, Fred H. Wilt, Irit Sagi, Lia Addadi,Steve Weiner, and Pupa Gilbert, which appeared in issue 45,November 11, 2008, of Proc Natl Acad Sci USA (105:17362–17366; first published November 5, 2008; 10.1073�pnas.0806604105), the authors note that the author name PupaGilbert should have appeared as P. U. P. A. Gilbert. The authorline has been corrected online. In addition, in the authorcontributions footnote and in the Acknowledgments, the initialsP.G. should appear as P.U.P.A.G. The corrected author lineappears below.

Yael Politi, Rebecca A. Metzler, Mike Abrecht, BenjaminGilbert, Fred H. Wilt, Irit Sagi, Lia Addadi, Steve Weiner,and P. U. P. A. Gilbert

www.pnas.org�cgi�doi�10.1073�pnas.0811530106

Fig. 3. E2 rapidly and transiently induces the formation of silent synapsesthrough trafficking of GluR1 and NR1. (A and B) Time-lapse imaging ofneurons expressing GFP-GluR1. Cells were imaged for 60 min before and afteradministration of E2. Arrowheads indicate GFP-GluR1 in spine heads; arrowsindicate GFP-GluR1 in dendritic shaft. Dotted lines indicate neuron outline, asdetermined by Discosoma red fluorescent protein coexpression; asterisksshow transient emergence of novel spines upon E2 treatment. (Scale bars, 1�m.) (C) AMPAR mEPSCs after E2 treatment. Frequency and average ampli-tude of mEPSCs were measured; frequency, but not amplitude, of mEPSCs wassignificantly reduced at 30 min. *, P � 0.05; ***, P � 0.001.

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Transformation mechanism of amorphous calciumcarbonate into calcite in the sea urchin larval spiculeYael Politia, Rebecca A. Metzlerb, Mike Abrechtc, Benjamin Gilbertd, Fred H. Wilte, Irit Sagia, Lia Addadia,1,Steve Weinera,1, and P. U. P. A. Gilbertb,1,2

aDepartment of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel; bDepartment of Physics, University of Wisconsin,Madison, WI 53706; cSynchrotron Radiation Center, Stoughton, WI 53589; dEarth Science Division, Lawrence Berkeley National Laboratory,Berkeley, CA 94720; and eDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200

Edited by Jack Halpern, University of Chicago, Chicago, IL, and approved September 25, 2008 (received for review July 8, 2008)

Sea urchin larval spicules transform amorphous calcium carbonate(ACC) into calcite single crystals. The mechanism of transformationis enigmatic: the transforming spicule displays both amorphousand crystalline properties, with no defined crystallization front.Here, we use X-ray photoelectron emission spectromicroscopywith probing size of 40–200 nm. We resolve 3 distinct mineralphases: An initial short-lived, presumably hydrated ACC phase,followed by an intermediate transient form of ACC, and finally thebiogenic crystalline calcite phase. The amorphous and crystallinephases are juxtaposed, often appearing in adjacent sites at a scaleof tens of nanometers. We propose that the amorphous-crystaltransformation propagates in a tortuous path through preexisting40- to 100-nm amorphous units, via a secondary nucleationmechanism.

biomineralization � Ca L-edge X-ray absorption near-edge structure �XANES � X-PEEM � X-ray photoelectron emission spectromicroscopy

A widespread strategy in biomineralization is the initialformation of transient amorphous precursor phases thatsubsequently transform into one of the more stable crystallinephases (1). This process was first observed in the teeth of chitonswhere a disordered ferrihydrite precursor transforms into mag-netite (2). It has also been observed in different invertebratephyla (3–8). Amorphous calcium phosphate was recently iden-tified in the newly deposited fin bones of zebrafish (9). Themechanistic details of these transformations are, however, stillpoorly understood. Here, we address this fundamental issue bystudying the transformation of amorphous calcium carbonate(ACC) to crystalline calcite in the sea urchin larval spicule. Seaurchin larval spicules have long served as a model system for thestudy of CaCO3 biomineralization processes, and the transientACC precursor phase was first identified in this system (3). Themature larval spicule is composed of a single crystal of magne-sium-bearing calcite (10, 11). Small amounts of organic macro-molecules (0.1 wt%) are incorporated within the mineral and areknown to play a role in the transient stabilization of theamorphous phase (12).

The spicules are formed inside a syncytium produced byspecialized cells (13). The first deposit is a single rhombohedral-shaped calcite crystal. Further growth of the spicule radii followscrystallographic orientations dictated by the initial crystal (10,14), even though the mineral deposited is mainly in the form ofACC. The rays elongate rapidly for �3 days, while the existingrays thicken. ACC is most probably introduced into the miner-alization site by the cells in vesicles that fuse with the syncytialmembrane (15). The spicule is tightly surrounded by this mem-brane, with no interstitial water solution detectable at any stage(16). In the polarized light microscope almost the entire spiculebehaves as a homogeneously bright birefringent domain, despitebeing composed mainly of an amorphous phase. The exceptionsare the growing tips of the spicule that show no birefringence,suggesting that they are completely amorphous (16). Partialdemineralization (etching) of the spicule shows that the spicule

is composed of densely packed mineral spherules 40–100 nmin diameter (3, 17). No crystallization front can be detected atthe micrometer scale. Extended X-ray absorption fine structure(EXAFS) spectroscopy at the Ca K-edge showed that even atearly stages, when the mineral is still predominantly amorphous,it already has a nascent short-range order around the calciumions similar to that in calcite (18). In contrast to stable biogenicACC, which contains 1 water molecule per CaCO3, the amor-phous phase in the spicules is mostly anhydrous when the spiculesare extracted at an advanced developmental stage (12, 19).Macroscopically, therefore, the spicule displays both amorphousand crystalline qualities.

Results and DiscussionUnraveling the mechanistic complexities of the spatial andtemporal interplay between the transforming amorphous andcrystalline phases requires the use of high-resolution techniques.Here, we use X-ray photoelectron emission spectromicroscopy(X-PEEM) to study the transformation at high spatial resolution(20, 21). We analyze X-ray absorption near-edge structure(XANES) (22) spectra at the Ca L-edge along the length ofspicules at two developmental stages. Ca spectra were acquiredby recording 170 images, 0.1 eV apart, and arranging them instacks in which the energy-dependent intensity of each pixelholds the full spectral information across the Ca L-edge. Thepixel size depends on the magnification and is 40–200 nm in thisstudy, while the probing depth is �3 nm at the Ca L-edge energyrange (23). This technique offers the unique opportunity ofcharacterizing the atomic order of the mineral phase (24) alonga single larval spicule with sub-micrometer spatial resolution,providing time and space-resolved snapshots of the crystalliza-tion pathway through 2 distinct amorphous phases.

Fig. 1 shows spectra acquired from a 48-h embryo spicule witha pixel size of 200 nm. At this stage the spicule is at the triradiatestage of development and is composed of 70–90% ACC (18).The spectra were extracted from areas near the tip and along 1of the spicule radii. The spectra near the tip are more hetero-geneous than those from the rest of the spicule, revealing thatnewly-formed regions of the spicule are structurally diverse.Similar results on a different 48-h spicule are presented insupporting information (SI) Fig. S1. For comparison, spectra

Author contributions: Y.P., I.S., L.A., and S.W. designed research; Y.P., R.A.M., M.A., F.H.W.,and P.G. performed research; M.A., B.G., F.H.W., and P.G. contributed new reagents/analytic tools; Y.P., R.A.M., B.G., I.S., L.A., S.W., and P.G. analyzed data; and Y.P., L.A., S.W.,and P.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1To whom correspondence may be addressed. E-mail: lia.addadi@weizmann.ac.il, steve.weiner@weizmann.ac.il, or pupa@physics.wisc.edu.

2Previously published as Gelsomina De Stasio.

This article contains supporting information online at www.pnas.org/cgi/content/full/0806604105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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from synthetic calcite and synthetic ACC are also shown in Fig.1 D and E, respectively. In calcite, the 2 main peaks (denoted 1and 3, which are the Ca L2 and L3 peaks, respectively) are split,giving rise to 2 minor peaks [denoted as 2 and 4, the crystal fieldpeaks (25)]. In synthetic ACC, peaks 2 and 4 are less intense andshifted closer to the main peaks, where they appear as shoulders.

Analysis of numerous single pixel spectra revealed 3 indepen-dent calcium absorption line-shapes that correspond to 3 differ-ent mineral phases (Fig. 1). The first spectrum type (red in Fig.1) is similar to synthetic ACC, where both peaks 2 and 4 areweak. This type 1 spectrum is found only near the tips of 48-hspicules. The second type (green in Fig. 1) has a pronouncedpeak 2 but a weak peak 4. The third type of spectrum (blue inFig. 1) is similar to that of calcite, with both peaks 2 and 4 beingintense. This third type of spectrum resembles calcite and isfound at locations everywhere on the spicule surface, but withgreater frequency and intensity in pixels at the center of thetriradiate structure, where the initial rhombohedral calcite crys-tal was observed (10) (Fig. S1). All other spectra fall betweenthese 3 types. The type 2 spectrum is distinct from those ofsynthetic ACC, and calcite spectra and cannot be the result of alinear combination of types 1 and 3, because any linear combi-nation of these leads to a parallel change in both peaks 2 and 4(Fig. S2). The suppression of the L3 crystal field peak 4 in type2 indicates that this spectrum represents a disordered form ofcalcium carbonate, although no similar spectrum has beenrecorded from standard materials. In both 48- and 72-h spicules,the most abundant phase is type 2.

Over time, the amorphous material present in fresh spicules isknown to crystallize (3), and we sought to determine whether theACC phases found here in fresh spicules, 48 h after fertilization,are transient phases. We repeated the measurements on freshspicules grown for a longer period (72 h after fertilization) andthen at precisely the same location on the same spicules after 10months in storage (Fig. 2 and Fig. S3). The fresh 72-h spicules

consist only of type 2 ACC, crystalline biogenic calcite, orcombinations thereof, with no type 1 ACC phase (Fig. 2E),whereas the spectra obtained from the 10-month-old spicule areuniformly similar to crystalline biogenic calcite (Fig. 2F). Thisresult implies that the type 2 phase is less stable than calcite. Weinfer that the crystalline phase grew at the expense of amorphousdomains and/or the amorphous phase, having higher solubility,was preferentially removed. The 10-month-old spicule has anetched appearance when imaged in the scanning electron mi-croscope (SEM) (Fig. 2D), resembling an aggregate of spheresof 40–100 nm in diameter. The etched spicule exhibits topo-graphic features that are coarser than nonetched spicules. Thehomogeneity of the spectra extracted from this sample thusdemonstrates that specimen topography cannot produce thespectral diversity shown in Fig. 2E.

Selected spectra from the 48- and 72-h spicules in Figs. 1 and2 and from the reference standards were peak-fitted. Thepeak-fitting results, presented in Fig. S4, highlight the spectro-scopic differences among type 1, 2, and 3 mineral phases, and thesimilarity of types 1 and 3 with synthetic ACC and calcite.

We further characterized the occurrence of these distinctcarbonate phases in 72-h spicules by repeating the measurementat higher magnification (40-nm pixels vs. 200- and 100-nm pixelsin Figs. 1 and 2) to gain more insight into the dimensions of theindividual domains. Indeed, isolated spectra of amorphous do-mains can be detected in the middle of crystalline domains. Weobserved abrupt transitions between calcite and ACC in imme-diately adjacent pixels, as well as more gradual transitions (Fig.3). The smallest domains observed are 1 pixel wide, and othersare 3 pixels wide (40–120 nm).

Because the phases may be distinguished by the crystal fieldsplitting at the L2 and L3 edges, we define an empirical splittingratio (SR) to be the ratio of the maximum intensity of thecrystal field peak (2 or 4) and the intensity of the minimumseparating this peak from the corresponding main peak (1 or 3)

Fig. 1. Ca L-edge XANES spectra and an X-PEEM micrograph of a 48-h spicule. (A and B) Ca L-edge XANES spectra extracted from: near the tip (left yellow linein C) (A) and middle part of the spicule (right yellow line in C) (B). (C) X-PEEM micrograph of part of a fresh 48-h spicule. (D) Ca L-edge XANES spectra of syntheticcalcite; the L2 peak is split into peaks 1 and 2, and the L3 is split into peaks 3 and 4. The main peaks are 1 and 3, and the crystal field peaks are 2 and 4. (E) CaL-edge XANES spectra of synthetic ACC. These and all spectra hereafter were extracted from adjacent pixels along a line. The bold spectra at the top of A, B, D,and E are the averages of all spectra below. Blue in A and B highlights a spectrum similar to calcite. Green highlights a spectrum with intense peak 2 and smallpeak 4. Red, present in A but not in B, highlights a spectrum similar to synthetic hydrated ACC. Each spectrum in A, B, D, and E was extracted from a 200-nm pixel.

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(Fig. 4 and Fig. S5). For synthetic ACC, both peaks are poorly splitand thus both SR values are less than unity. For biogenic andsynthetic calcite, both peaks are well resolved, and the SRs arelarger than 2 and 1.3, for L2 and L3, respectively. The single-pixelSR values for spicules are varied, and structural trends in the datafrom the biogenic samples are illustrated by plotting L3 SR vs. L2

SR (Fig. 4B). Each of the 3 phase types identified above falls in adifferent quadrant of this plot (Fig. 4C): ACC type 1 with bothSRs � 1; ACC type 2, with L2 SR � 1 and L3 SR � 1; and calciticwith both SRs � 1.

The spectra are often a mixture of phases. This mixture occurswhen a pixel includes, for instance, part of a type 2 and part ofa type 3 particle. Thus the corresponding spectrum and SRs areintermediate between types 2 and 3. The spicule SRs tend tobecome more calcite-like with increasing distance from spiculetip to the middle, and with growth time from 48 to 72 h afterfertilization (Fig. 4). All SR values obtained from the 72-hspicule after 10 months fall in the calcite-like top-right quadrant,as expected. However, the values are much smaller than those ofsynthetic calcite, indicating greater structural disorder. Thisresult might be attributed to the presence in the spicules of �5mole% magnesium and the occluded matrix proteins. To supportthis hypothesis, we measured the SRs of adult sea urchin spines,considered to be composed of crystalline calcite exclusively. Thespine’s SRs (Fig. 4B), with Mg concentration similar to thespicules, also do not reach the values of synthetic calcite,although they are shifted toward calcite relative to the spicule.Consistently, spectra of biogenic minerals containing higheramounts of Mg have even lower SRs, but with both SRs � 1(Yurong Ma, personal communication).

The present data thus show that spicule development involves2 amorphous precursor phases. The freshly deposited mineral issimilar to hydrated synthetic ACC. This phase is short-lived andcan be detected only in areas of fast growth (the tip), rather thanwhere slower radial thickening occurs (17). This type 1 ACCrapidly transforms into a second phase that appears amorphousfrom the XANES data. This type 2 ACC transforms more slowlyinto biogenic calcite.

Prior studies on spicules extracted at an advanced develop-mental stage have shown that the amorphous phase in thissystem is mostly anhydrous (19). However, it has been suggested

A

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Fig. 2. X-PEEM micrographs and Ca L-edge XANES spectra of a fresh 72-h spicule, and the same spicule after 10 months. (A–C) X-PEEM micrographs of the 72-hspicule. (A) Fresh spicule: the dark region immediately below the spicule is its shadow. (B) The same spicule measured 10 months later. The yellow lines indicatethe pixels from which the spectra in E and F were extracted. (C) High magnification of the area in A: colored pixels are those from which the corresponding coloredspectra in E and F were extracted. (D) SEM micrograph of the same spicule taken after 10 months. (E and F) Ca L-edge spectra extracted from the lines in A andB, respectively. The blue curve in E corresponds to the type 2 ACC phase, which clearly became calcitic in F. Scale bar in A also applies to B, and scale bar in C alsoapplies to D. Pixel size is 100 � 100 nm2. Highlighted in black is the average spectrum.

Fig. 3. Ca L-edge XANES spectra from a fresh 72-h spicule. The spectra areextracted from individual pixels along a straight line. Each pixel represents40 � 40 nm2. The 2 series of spectra show different patterns of mineral phasedistribution in adjacent pixels. (A) We observe large blocks of 5–10 adjacentspectra of type 3 calcite interspersed with smaller series of spectra of type 2 ACC.(B) The transitions between type 2 and 3 spectra are abrupt between the bottom2 spectraandgradual fortheothers.Highlightedinblackaretheaveragespectra.Blue highlights 1 of the type 3 calcite spectra, green highlights type 2 ACC.

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that water molecules may be present as part of an initial hydratedACC phase that subsequently transforms into the anhydrousphase (7, 18). Our data are in good agreement with thismechanism, and we suggest that they represent direct observa-tion of the dehydration step for freshly deposited ACC. Becausethis type 2 phase is the most abundant in fresh spicules it is likelyto be the same anhydrous ACC phase observed with bulkmethods (18, 19) and now confirmed to be formed from anearlier transient phase. We note that the probing depth in theseexperiments is only 3 nm. Thus only the fresh material that isdeposited on the spicule surface upon thickening is sampled.

A recent EXAFS study showed that the transient ACC phaseat this stage has a short-range order that resembles the maturecrystalline phase (18), which might be the origin of the promi-nent peak 2. The type 2 mineral is therefore intermediatebetween fully disordered, probably hydrated ACC and crystal-line calcite with respect to both spicule development stage andcrystallinity.

The present data show the presence of juxtaposed crystallineand amorphous phases in the surface layer of growing spicules,raising the fundamental question of how these heterogeneousmineral domains transform into the single crystal found in themature spicule. Insight is obtained from the higher-resolutionanalysis of the size of single domains. In all specimens, weobserve discontinuity in mineral phases in immediately adjacentpixels, suggesting that the precursor mineral phase is present insmall units. Analysis of 72-h specimens showed that homoge-neous domains of type 3 calcite as large as 1 �m are present,interspersed with smaller domains of type 2 ACC. The smallestACC domains (40–120 nm) observed here with X-PEEM, areconsistent in size with the previous observation of 50–100 nmspherules (Fig. 2) (3, 17) and the coherence length of the maturespicule observed in X-ray diffraction (26).

From these observations, we propose that a transformationfrom type 2 amorphous to type 3 crystalline phase propagatesthrough the spicule via secondary nucleation, in which thecrystallization of 1 amorphous unit stimulates the transforma-tion of the domains in contact with it (27, 28). The overallcrystallographic orientation is determined by the initial centralcrystal. Because type 2 ACC is anhydrous, no volume changeoccurs during the transformation to type 3 calcite, and spiculemorphology is unaffected.

The propagation pathway through the spicule is inferred to becomplex and tortuous, implying that the rate of transformationdepends on the size and interface structure of the amorphousdomains. These are probably determined by the presence, loca-tion, and concentration of the organic additives. Within largercalcitic regions of 72-h spicules, individual amorphous domainsof 40 � 40 nm2 were occasionally identified, indicating that thepropagation pathway may leave small domains untransformed.Mapping the distribution of single-phase domains, as demon-strated here, will enable the testing of various hypotheses thatmay account for the patterns of crystalline-phase propagationthat are akin to fractal network percolation (29, 30).

The transformation mechanism presented here may wellrepresent a common strategy in biomineralization, bearing inmind the widespread use of precursor ACC phases in biology andthe many cases in which 30- to 50-nm spherulitic structures havebeen observed in biogenic calcium carbonate minerals fromdiverse phyla (31–35).

MethodsMore detailed descriptions of the experimental procedures are provided in SIText.

Sea Urchin Larval Culture. Strongylocentrotus purpuratus embryos weregrown in artificial sea water containing Gentamycin (20 mg�L�1) at 15 °C,following established methods (36, 37).

Extraction of S. purpuratus Spicules. Embryos were disrupted in a Polytronhomogenizer. The spicules were collected by centrifugation and extractedwith SDS and 3.5% NaOCl. The spicules were washed with CaCO3-saturatedsolution and rinsed with ethanol and acetone, frozen in liquid nitrogen andkept at �80 °C for up to 2 days until the measurements.

Synthetic calcite crystals were grown in Nunc multiwell dishes by diffusionof ammonium carbonate vapor into 10 mM calcium chloride (Merck; A grade)solutions (38). Synthetic ACC was synthesized following Koga et al. (39) bymixing solutions of calcium chloride (0.1 M) with sodium carbonate (0.1 M)and sodium hydroxide (1 M).

X-PEEM Sample Preparation. Forty-eight- or 72-h spicule samples were resus-pended in ethanol. A drop of the suspension was deposited on a 10 mm �10 mm silicon chip and air-dried. Synthetic ACC and calcite powders werepressed into indium foil. All samples were sputter-coated with 1 nm Pt (40).

X-PEEM Experiments. We used the spectromicroscope for photoelectron im-aging of nanostructures with X-rays (SPHINX), which is an X-PEEM (Elmitec),installed on the VLS-PGM beamline at the Synchrotron Radiation Center. Theinstrument and its performances are described in detail in ref. 20. Briefly, thesample was mounted vertically and illuminated from a side (16° grazingincidence angle) with monochromatic soft X-rays. The photoelectrons emittedby the sample were accelerated toward an electron optics column and onto aphosphor screen. The chamber was held at ultrahigh vacuum (10�10 Torr). Thereal-time, sample surface image was acquired by a slow-scanning cooled CCDcamera. Movie stacks of 170 images were acquired while scanning the photonenergy across the Ca L-absorption edge, so that each pixel in the movie containeda complete XANES spectrum. Images were acquired with fields of view rangingfrom 20 to 100 �m and corresponding pixel sizes of 40–200 nm. Because thesamples are not flat, the spatial resolution may be lower than the pixel size.

Data Analysis. Data processing was done by using routines we designed inNational Institutes of Health Image 1.62 and Igor Pro 6.0.3 (WaveMetrics) forMacintosh. From each stack of 170 images Ca L-edge spectra were extractedfrom single pixels along a straight line on the spicule or standards and divided

Fig. 4. Peak splitting anaylsis of Ca L-edge XANES spectra from 48-h and 72-hspicules, as well as those from ACC and calcite. (A) Ca L-edge XANES spectraextracted from single pixels of synthetic ACC (bottom red curve) and calcite(top blue curve) and 3 spectra from a 48-h spicule. The 3 spicule spectra arerepresentative of the 3 mineral phases identified in Fig. 1: red is type 1, greenis type 2, and blue is type 3. (B) A plot of SR(L3) vs. SR(L2) (see Methods and Fig.S5). The spicule samples indicated by triangles (48-h spicule tip, light blue;middle, purple; 72-h spicule fresh, green; 10 month olds, blue), and the adult seaurchin spine (squares, brown), span 3 quadrants, and synthetic ACC (red circles) islocated in the bottom left quadrant, where L2, L3 SR � 1. Calcite (blue diamonds)is locatedinthetoprightquadrantwhereL2,L3 SR�1. (C)Spiculeratiosareshownseparately with the relevant quadrants shaded in gray. Color code is as in B.

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by a pre-edge linear background. All spectra presented here were normalizedto a linear pre-edge fit. The position of peak 1 was set to be 352.6 eV for allspectra, following Benzerara et al. (41). The intensity of the pre-edge was thenset to 0 and that of peak 1 to 1. All spectra were smoothed over 5 points anddisplaced vertically in all figures.

Peak Fitting. Selected spectra were peak-fitted by using routines we designedin Igor Pro 6.0.3 (WaveMetrics). The most representative peak-fitted spectraare presented in Fig. S4.

Calculations of SRs. For each peak we divided the intensity value, afterapproximated-baseline subtraction, of the split peak (spL2, spL3) by the inten-sity value of the minimum between these peaks and the main peak (dipL2 anddipL3, respectively). Such that:

SRL2 � spL2/dipL2

SRL3 � spL3/dipL3.

See Fig. S5.

Scanning Electron Microscopy. Samples were coated with 6 nm Cr and viewedwith a SEM (Philips; XL30 FESEM FEG), operated at 10 kV.

ACKNOWLEDGMENTS. We thank Prof. Peter Rez for fruitful discussions. Thiswork was supported by National Science Foundation Award CHE&DMR-0613972 (to P.G.), Department of Energy Award DE-FG02-07ER15899 (to P.G.and S.W.), and Israel Ministry of Science Project 777. The experiments wereperformed at the University of Wisconsin–Synchrotron Radiation Center,which was supported by National Science Foundation Award DMR-0537588.F.H.W. is supported by the National Institutes of Health and National ScienceFoundation. L.A. is the incumbent of the Dorothy and Patrick Gorman Pro-fessorial Chair of Biological Ultrastructure, and S.W. is the incumbent of theDr. Walter and Dr. Trude Burchardt Professorial Chair of Structural Biology. I.S.is the incumbent of the Pontecorvo Professorial Chair of Cancer Research.

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