Transformation Mechanism of Amorphous Calcium Carbonate Into Calcite in the Sea Urchin Larval Spicule

NEUROSCIENCE. For the article ‘‘Rapid enhancement of two-step APPLIED BIOLOGICAL SCIENCES. For the article ‘‘Accurately quanti- wiring plasticity by estrogen and NMDA receptor activity,’’ by fying low-abundant targets amid similar sequences by revealing Deepak P. Srivastava, Kevin Woolfrey, Kelly A. Jones, Cassan- hidden correlations in oligonucleotide microarray data,’’ by dra Y. Shum, L. Leanne Lash, Geoffrey T. Swanson, and Peter Luisa A. Marcelino, Vadim Backman, Andres Donaldson, Clau- Penzes, which appeared in issue 38, September 23, 2008, of Proc dia Steadman, Janelle R. Thompson, Sarah Pacocha Preheim, Natl Acad Sci USA (105:14650–14655; first published September Cynthia Lien, Eelin Lim, Daniele Veneziano, and Martin F. 18, 2008; 10.1073͞pnas.0801581105), the authors note that the Polz, which appeared in issue 37, September 12, 2006, of Proc author name Kevin Woolfrey should have appeared as Kevin M. Natl Acad Sci USA (103:13629–13634; first published September Woolfrey. The author line has been corrected online. In addition, 1, 2006; 10.1073͞pnas.0601476103), the authors note that in Eq. in the author contributions footnote and in the Acknowledg- 4,aϪ1 was inadvertently omitted from the denominator. The ments, the initials K.W. should appear as K.M.W. The authors data in Fig. 1 were calculated using the correct equation, and this also note that due to a printer’s error, in Fig. 3A, some colors error in the published equation would make Ͻ1% difference in printed incorrectly. The corrected author line, and the corrected the values of ␤. The corrected equation appears below. figure and its legend, appear below. 1 1 Deepak P. Srivastava, Kevin M. Woolfrey, Kelly A. Jones, ␤ ϭ Ϫ jk 1 ⌬ , [4] b Gjk Cassandra Y. Shum, L. Leanne Lash, Geoffrey T. Swanson, ΂ Ϫ2 hͩ Ϫ1ͪ΃ ͱ ϩ ͩ͑ Ϫ ͒ Ϫ ͪ ⌬ 1 1 b 1 e Gjj and Peter Penzes

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BIOPHYSICS, CHEMISTRY. For the article ‘‘Transformation mechanism of amorphous calcium carbonate into calcite in the sea urchin larval spicule,’’ by Yael Politi, Rebecca A. Metzler, Mike Abrecht, 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; 10.1073͞pnas. 0806604105), the authors note that the author name Pupa Gilbert should have appeared as P. U. P. A. Gilbert. The author line has been corrected online. In addition, in the author contributions footnote and in the Acknowledgments, the initials P.G. should appear as P.U.P.A.G. The corrected author line appears below. Yael Politi, Rebecca A. Metzler, Mike Abrecht, Benjamin Gilbert, Fred H. Wilt, Irit Sagi, Lia Addadi, Steve Weiner, and P. U. P. A. Gilbert

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Fig. 3. E2 rapidly and transiently induces the formation of silent synapses through trafficking of GluR1 and NR1. (A and B) Time-lapse imaging of neurons expressing GFP-GluR1. Cells were imaged for 60 min before and after administration of E2. Arrowheads indicate GFP-GluR1 in spine heads; arrows indicate GFP-GluR1 in dendritic shaft. Dotted lines indicate neuron outline, as determined by Discosoma red fluorescent protein coexpression; asterisks show transient emergence of novel spines upon E2 treatment. (Scale bars, 1 ␮m.) (C) AMPAR mEPSCs after E2 treatment. Frequency and average amplitude of mEPSCs were measured; frequency, but not amplitude, of mEPSCs was significantly reduced at 30 min. *, P Ͻ 0.05; ***, P Ͻ 0.001.

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PNAS ͉ December 16, 2008 ͉ vol. 105 ͉ no. 50 ͉ 20045 Downloaded by guest on September 25, 2021 Transformation mechanism of amorphous calcium carbonate into calcite in the sea urchin larval spicule

Yael 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 is composed of densely packed mineral spherules 40–100 nm (ACC) into calcite single crystals. The mechanism of transformation in diameter (3, 17). No crystallization front can be detected at is enigmatic: the transforming spicule displays both amorphous the micrometer scale. Extended X-ray absorption fine structure and crystalline properties, with no defined crystallization front. (EXAFS) spectroscopy at the Ca K-edge showed that even at Here, we use X-ray photoelectron emission spectromicroscopy early stages, when the mineral is still predominantly amorphous, with probing size of 40–200 nm. We resolve 3 distinct mineral it already has a nascent short-range order around the calcium phases: An initial short-lived, presumably hydrated ACC phase, ions similar to that in calcite (18). In contrast to stable biogenic followed by an intermediate transient form of ACC, and finally the ACC, which contains 1 water molecule per CaCO3, the amor- biogenic crystalline calcite phase. The amorphous and crystalline phous phase in the spicules is mostly anhydrous when the spicules phases are juxtaposed, often appearing in adjacent sites at a scale are extracted at an advanced developmental stage (12, 19). of tens of nanometers. We propose that the amorphous-crystal Macroscopically, therefore, the spicule displays both amorphous transformation propagates in a tortuous path through preexisting and crystalline qualities. 40- to 100-nm amorphous units, via a secondary nucleation mechanism. Results and Discussion Unraveling the mechanistic complexities of the spatial and biomineralization ͉ Ca L-edge X-ray absorption near-edge structure ͉ temporal interplay between the transforming amorphous and XANES ͉ X-PEEM ͉ X-ray photoelectron emission spectromicroscopy crystalline phases requires the use of high-resolution techniques. Here, we use X-ray photoelectron emission spectromicroscopy widespread strategy in biomineralization is the initial (X-PEEM) to study the transformation at high spatial resolution Aformation of transient amorphous precursor phases that (20, 21). We analyze X-ray absorption near-edge structure subsequently transform into one of the more stable crystalline (XANES) (22) spectra at the Ca L-edge along the length of phases (1). This process was first observed in the teeth of chitons spicules at two developmental stages. Ca spectra were acquired where a disordered ferrihydrite precursor transforms into mag- by recording 170 images, 0.1 eV apart, and arranging them in netite (2). It has also been observed in different invertebrate stacks in which the energy-dependent intensity of each pixel phyla (3–8). Amorphous calcium phosphate was recently iden- holds the full spectral information across the Ca L-edge. The tified in the newly deposited fin bones of zebrafish (9). The pixel size depends on the magnification and is 40–200 nm in this mechanistic details of these transformations are, however, still study, while the probing depth is Ϸ3nmattheCaL-edge energy poorly understood. Here, we address this fundamental issue by range (23). This technique offers the unique opportunity of studying the transformation of amorphous calcium carbonate characterizing the atomic order of the mineral phase (24) along (ACC) to crystalline calcite in the sea urchin larval spicule. Sea a single larval spicule with sub-micrometer spatial resolution, urchin larval spicules have long served as a model system for the providing time and space-resolved snapshots of the crystalliza- study of CaCO3 biomineralization processes, and the transient tion pathway through 2 distinct amorphous phases. ACC precursor phase was first identified in this system (3). The Fig. 1 shows spectra acquired from a 48-h embryo spicule with mature larval spicule is composed of a single crystal of magne- a pixel size of 200 nm. At this stage the spicule is at the triradiate sium-bearing calcite (10, 11). Small amounts of organic macro- stage of development and is composed of 70–90% ACC (18). molecules (0.1 wt%) are incorporated within the mineral and are The spectra were extracted from areas near the tip and along 1 known to play a role in the transient stabilization of the of the spicule radii. The spectra near the tip are more hetero- amorphous phase (12). geneous than those from the rest of the spicule, revealing that The spicules are formed inside a syncytium produced by newly-formed regions of the spicule are structurally diverse. specialized cells (13). The first deposit is a single rhombohedral- Similar results on a different 48-h spicule are presented in shaped calcite crystal. Further growth of the spicule radii follows supporting information (SI) Fig. S1. For comparison, spectra crystallographic orientations dictated by the initial crystal (10, 14), even though the mineral deposited is mainly in the form of Ϸ Author contributions: Y.P., I.S., L.A., and S.W. designed research; Y.P., R.A.M., M.A., F.H.W., ACC. The rays elongate rapidly for 3 days, while the existing and P.G. performed research; M.A., B.G., F.H.W., and P.G. contributed new reagents/ rays thicken. ACC is most probably introduced into the miner- 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., alization site by the cells in vesicles that fuse with the syncytial and P.G. wrote the paper. membrane (15). The spicule is tightly surrounded by this mem- The authors declare no conflict of interest. brane, with no interstitial water solution detectable at any stage This article is a PNAS Direct Submission. (16). In the polarized light microscope almost the entire spicule 1To whom correspondence may be addressed. E-mail: [email protected], steve. behaves as a homogeneously bright birefringent domain, despite [email protected], or [email protected]. being composed mainly of an amorphous phase. The exceptions 2Previously published as Gelsomina De Stasio. are the growing tips of the spicule that show no birefringence, This article contains supporting information online at www.pnas.org/cgi/content/full/ suggesting that they are completely amorphous (16). Partial 0806604105/DCSupplemental. demineralization (etching) of the spicule shows that the spicule © 2008 by The National Academy of Sciences of the USA

17362–17366 ͉ PNAS ͉ November 11, 2008 ͉ vol. 105 ͉ no. 45 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806604105 Fig. 1. Ca L-edge XANES spectra and an X-PEEM micrograph of a 48-h spicule. (A and B)CaL-edge XANES spectra extracted from: near the tip (left yellow line in 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)CaL-edge XANES spectra of synthetic calcite; 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)Ca L-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 small peak 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. from synthetic calcite and synthetic ACC are also shown in Fig. consist only of type 2 ACC, crystalline biogenic calcite, or 1 D and E, respectively. In calcite, the 2 main peaks (denoted 1 combinations thereof, with no type 1 ACC phase (Fig. 2E), CHEMISTRY and 3, which are the Ca L2 and L3 peaks, respectively) are split, whereas the spectra obtained from the 10-month-old spicule are giving rise to 2 minor peaks [denoted as 2 and 4, the crystal field uniformly similar to crystalline biogenic calcite (Fig. 2F). This peaks (25)]. In synthetic ACC, peaks 2 and 4 are less intense and result implies that the type 2 phase is less stable than calcite. We shifted closer to the main peaks, where they appear as shoulders. infer that the crystalline phase grew at the expense of amorphous Analysis of numerous single pixel spectra revealed 3 indepen- domains and/or the amorphous phase, having higher solubility, dent calcium absorption line-shapes that correspond to 3 differ- was preferentially removed. The 10-month-old spicule has an ent mineral phases (Fig. 1). The first spectrum type (red in Fig. etched appearance when imaged in the scanning electron mi- 1) is similar to synthetic ACC, where both peaks 2 and 4 are croscope (SEM) (Fig. 2D), resembling an aggregate of spheres BIOPHYSICS weak. This type 1 spectrum is found only near the tips of 48-h of 40–100 nm in diameter. The etched spicule exhibits topo- spicules. The second type (green in Fig. 1) has a pronounced graphic features that are coarser than nonetched spicules. The peak 2 but a weak peak 4. The third type of spectrum (blue in homogeneity of the spectra extracted from this sample thus Fig. 1) is similar to that of calcite, with both peaks 2 and 4 being demonstrates that specimen topography cannot produce the intense. This third type of spectrum resembles calcite and is spectral diversity shown in Fig. 2E. found at locations everywhere on the spicule surface, but with Selected spectra from the 48- and 72-h spicules in Figs. 1 and greater frequency and intensity in pixels at the center of the 2 and from the reference standards were peak-fitted. The triradiate structure, where the initial rhombohedral calcite crys- peak-fitting results, presented in Fig. S4, highlight the spectro- tal was observed (10) (Fig. S1). All other spectra fall between scopic differences among type 1, 2, and 3 mineral phases, and the these 3 types. The type 2 spectrum is distinct from those of similarity of types 1 and 3 with synthetic ACC and calcite. synthetic ACC, and calcite spectra and cannot be the result of a We further characterized the occurrence of these distinct linear combination of types 1 and 3, because any linear combi- carbonate phases in 72-h spicules by repeating the measurement nation of these leads to a parallel change in both peaks 2 and 4 at higher magnification (40-nm pixels vs. 200- and 100-nm pixels (Fig. S2). The suppression of the L3 crystal field peak 4 in type in Figs. 1 and 2) to gain more insight into the dimensions of the 2 indicates that this spectrum represents a disordered form of individual domains. Indeed, isolated spectra of amorphous do- calcium carbonate, although no similar spectrum has been mains can be detected in the middle of crystalline domains. We recorded from standard materials. In both 48- and 72-h spicules, observed abrupt transitions between calcite and ACC in imme- the most abundant phase is type 2. diately adjacent pixels, as well as more gradual transitions (Fig. Over time, the amorphous material present in fresh spicules is 3). The smallest domains observed are 1 pixel wide, and others known to crystallize (3), and we sought to determine whether the are 3 pixels wide (40–120 nm). ACC phases found here in fresh spicules, 48 h after fertilization, Because the phases may be distinguished by the crystal field are transient phases. We repeated the measurements on fresh splitting at the L2 and L3 edges, we define an empirical splitting spicules grown for a longer period (72 h after fertilization) and ratio (SR) to be the ratio of the maximum intensity of the then at precisely the same location on the same spicules after 10 crystal field peak (2 or 4) and the intensity of the minimum months in storage (Fig. 2 and Fig. S3). The fresh 72-h spicules separating this peak from the corresponding main peak (1 or 3)

Politi et al. PNAS ͉ November 11, 2008 ͉ vol. 105 ͉ no. 45 ͉ 17363 A E F

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-h spicule. (A) Fresh spicule: the dark region immediately below the spicule is its shadow. (B) The same spicule measured 10 months later. The yellow lines indicate the 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 colored spectra in E and F were extracted. (D) SEM micrograph of the same spicule taken after 10 months. (E and F)CaL-edge spectra extracted from the lines in A and B, 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 also applies to D. Pixel size is 100 ϫ 100 nm2. Highlighted in black is the average spectrum.

(Fig. 4 and Fig. S5). For synthetic ACC, both peaks are poorly split SR (Fig. 4B). Each of the 3 phase types identified above falls in a and thus both SR values are less than unity. For biogenic and different quadrant of this plot (Fig. 4C): ACC type 1 with both synthetic calcite, both peaks are well resolved, and the SRs are SRs Ͻ 1; ACC type 2, with L2 SR Ͼ 1 and L3 SR Ͻ 1; and calcitic Ͼ larger than 2 and 1.3, for L2 and L3, respectively. The single-pixel with both SRs 1. SR values for spicules are varied, and structural trends in the data The spectra are often a mixture of phases. This mixture occurs from the biogenic samples are illustrated by plotting L3 SR vs. L2 when a pixel includes, for instance, part of a type 2 and part of a type 3 particle. Thus the corresponding spectrum and SRs are intermediate between types 2 and 3. The spicule SRs tend to become more calcite-like with increasing distance from spicule tip to the middle, and with growth time from 48 to 72 h after fertilization (Fig. 4). All SR values obtained from the 72-h spicule after 10 months fall in the calcite-like top-right quadrant, as expected. However, the values are much smaller than those of synthetic calcite, indicating greater structural disorder. This result might be attributed to the presence in the spicules of Ϸ5 mole% magnesium and the occluded matrix proteins. To support this hypothesis, we measured the SRs of adult sea urchin spines, considered to be composed of crystalline calcite exclusively. The spine’s SRs (Fig. 4B), with Mg concentration similar to the spicules, 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 higher amounts of Mg have even lower SRs, but with both SRs Ͼ 1 (Yurong Ma, personal communication). The present data thus show that spicule development involves 2 amorphous precursor phases. The freshly deposited mineral is similar to hydrated synthetic ACC. This phase is short-lived and can be detected only in areas of fast growth (the tip), rather than Fig. 3. Ca L-edge XANES spectra from a fresh 72-h spicule. The spectra are where slower radial thickening occurs (17). This type 1 ACC extracted from individual pixels along a straight line. Each pixel represents rapidly transforms into a second phase that appears amorphous ϫ 2 40 40 nm . The 2 series of spectra show different patterns of mineral phase from the XANES data. This type 2 ACC transforms more slowly distribution in adjacent pixels. (A) We observe large blocks of 5–10 adjacent spectra of type 3 calcite interspersed with smaller series of spectra of type 2 ACC. into biogenic calcite. (B) The transitions between type 2 and 3 spectra are abrupt between the bottom Prior studies on spicules extracted at an advanced develop- 2 spectra and gradual for the others. Highlighted in black are the average spectra. mental stage have shown that the amorphous phase in this Blue highlights 1 of the type 3 calcite spectra, green highlights type 2 ACC. system is mostly anhydrous (19). However, it has been suggested

17364 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806604105 Politi et al. From these observations, we propose that a transformation from type 2 amorphous to type 3 crystalline phase propagates through the spicule via secondary nucleation, in which the crystallization of 1 amorphous unit stimulates the transformation of the domains in contact with it (27, 28). The overall crystallographic orientation is determined by the initial central crystal. Because type 2 ACC is anhydrous, no volume change occurs during the transformation to type 3 calcite, and spicule morphology is unaffected. The propagation pathway through the spicule is inferred to be complex and tortuous, implying that the rate of transformation depends on the size and interface structure of the amorphous domains. These are probably determined by the presence, location, and concentration of the organic additives. Within larger calcitic regions of 72-h spicules, individual amorphous domains of 40 ϫ 40 nm2 were occasionally identified, indicating that the propagation pathway may leave small domains untransformed. Mapping the distribution of single-phase domains, as demon- strated here, will enable the testing of various hypotheses that may account for the patterns of crystalline-phase propagation that are akin to fractal network percolation (29, 30). Fig. 4. Peak splitting anaylsis of Ca L-edge XANES spectra from 48-h and 72-h The transformation mechanism presented here may well spicules, as well as those from ACC and calcite. (A)CaL-edge XANES spectra represent a common strategy in biomineralization, bearing in extracted from single pixels of synthetic ACC (bottom red curve) and calcite mind the widespread use of precursor ACC phases in biology and (top blue curve) and 3 spectra from a 48-h spicule. The 3 spicule spectra are the many cases in which 30- to 50-nm spherulitic structures have representative of the 3 mineral phases identiﬁed in Fig. 1: red is type 1, green been observed in biogenic calcium carbonate minerals from is 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; diverse phyla (31–35). middle, purple; 72-h spicule fresh, green; 10 month olds, blue), and the adult sea urchin spine (squares, brown), span 3 quadrants, and synthetic ACC (red circles) is Methods located in the bottom left quadrant, where L2, L3 SR Ͻ 1. Calcite (blue diamonds) More detailed descriptions of the experimental procedures are provided in SI is located in the top right quadrant where L2,L3 SR Ͼ1. (C) Spicule ratios are shown Text. separately with the relevant quadrants shaded in gray. Color code is as in B. Sea Urchin Larval Culture. Strongylocentrotus purpuratus embryos were grown in artiﬁcial sea water containing Gentamycin (20 mg⅐LϪ1) at 15 °C, following established methods (36, 37). that water molecules may be present as part of an initial hydrated CHEMISTRY ACC phase that subsequently transforms into the anhydrous phase (7, 18). Our data are in good agreement with this Extraction of S. purpuratus Spicules. Embryos were disrupted in a Polytron mechanism, and we suggest that they represent direct observa- homogenizer. The spicules were collected by centrifugation and extracted with SDS and 3.5% NaOCl. The spicules were washed with CaCO3-saturated tion of the dehydration step for freshly deposited ACC. Because solution and rinsed with ethanol and acetone, frozen in liquid nitrogen and this type 2 phase is the most abundant in fresh spicules it is likely kept at Ϫ80 °C for up to 2 days until the measurements. to be the same anhydrous ACC phase observed with bulk Synthetic calcite crystals were grown in Nunc multiwell dishes by diffusion methods (18, 19) and now confirmed to be formed from an of ammonium carbonate vapor into 10 mM calcium chloride (Merck; A grade)

earlier transient phase. We note that the probing depth in these solutions (38). Synthetic ACC was synthesized following Koga et al. (39) by BIOPHYSICS experiments is only 3 nm. Thus only the fresh material that is mixing solutions of calcium chloride (0.1 M) with sodium carbonate (0.1 M) deposited on the spicule surface upon thickening is sampled. and sodium hydroxide (1 M). A recent EXAFS study showed that the transient ACC phase X-PEEM Sample Preparation. Forty-eight- or 72-h spicule samples were resus- at this stage has a short-range order that resembles the mature pended in ethanol. A drop of the suspension was deposited on a 10 mm ϫ crystalline phase (18), which might be the origin of the promi- 10 mm silicon chip and air-dried. Synthetic ACC and calcite powders were nent peak 2. The type 2 mineral is therefore intermediate pressed into indium foil. All samples were sputter-coated with 1 nm Pt (40). between fully disordered, probably hydrated ACC and crystalline calcite with respect to both spicule development stage and X-PEEM Experiments. We used the spectromicroscope for photoelectron im- crystallinity. aging of nanostructures with X-rays (SPHINX), which is an X-PEEM (Elmitec), The present data show the presence of juxtaposed crystalline installed on the VLS-PGM beamline at the Synchrotron Radiation Center. The and amorphous phases in the surface layer of growing spicules, instrument and its performances are described in detail in ref. 20. Briefly, the raising the fundamental question of how these heterogeneous sample was mounted vertically and illuminated from a side (16° grazing incidence angle) with monochromatic soft X-rays. The photoelectrons emitted mineral domains transform into the single crystal found in the by the sample were accelerated toward an electron optics column and onto a mature spicule. Insight is obtained from the higher-resolution phosphor screen. The chamber was held at ultrahigh vacuum (10Ϫ10 Torr). The analysis of the size of single domains. In all specimens, we real-time, sample surface image was acquired by a slow-scanning cooled CCD observe discontinuity in mineral phases in immediately adjacent camera. Movie stacks of 170 images were acquired while scanning the photon pixels, suggesting that the precursor mineral phase is present in energy across the Ca L-absorption edge, so that each pixel in the movie contained small units. Analysis of 72-h specimens showed that homoge- a complete XANES spectrum. Images were acquired with fields of view ranging ␮ neous domains of type 3 calcite as large as 1 ␮m are present, from 20 to 100 m and corresponding pixel sizes of 40–200 nm. Because the samples are not flat, the spatial resolution may be lower than the pixel size. interspersed with smaller domains of type 2 ACC. The smallest ACC domains (40–120 nm) observed here with X-PEEM, are Data Analysis. Data processing was done by using routines we designed in consistent in size with the previous observation of 50–100 nm National Institutes of Health Image 1.62 and Igor Pro 6.0.3 (WaveMetrics) for spherules (Fig. 2) (3, 17) and the coherence length of the mature Macintosh. From each stack of 170 images Ca L-edge spectra were extracted spicule observed in X-ray diffraction (26). from single pixels along a straight line on the spicule or standards and divided

Politi et al. PNAS ͉ November 11, 2008 ͉ vol. 105 ͉ no. 45 ͉ 17365 by a pre-edge linear background. All spectra presented here were normalized ϭ SRL3 spL3/dipL3. to a linear pre-edge fit. The position of peak 1 was set to be 352.6 eV for all spectra, following Benzerara et al. (41). The intensity of the pre-edge was then See Fig. S5. set to 0 and that of peak 1 to 1. All spectra were smoothed over 5 points and displaced vertically in all figures. Scanning Electron Microscopy. Samples were coated with 6 nm Cr and viewed with a SEM (Philips; XL30 FESEM FEG), operated at 10 kV. Peak Fitting. Selected spectra were peak-fitted by using routines we designed in Igor Pro 6.0.3 (WaveMetrics). The most representative peak-fitted spectra ACKNOWLEDGMENTS. We thank Prof. Peter Rez for fruitful discussions. This are presented in Fig. S4. work 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 were Calculations of SRs. For each peak we divided the intensity value, after performed at the University of Wisconsin–Synchrotron Radiation Center, approximated-baseline subtraction, of the split peak (spL2,spL3) by the inten- which was supported by National Science Foundation Award DMR-0537588. sity value of the minimum between these peaks and the main peak (dipL2 and F.H.W. is supported by the National Institutes of Health and National Science dipL3, respectively). Such that: Foundation. L.A. is the incumbent of the Dorothy and Patrick Gorman Pro- fessorial Chair of Biological Ultrastructure, and S.W. is the incumbent of the ϭ Dr. Walter and Dr. Trude Burchardt Professorial Chair of Structural Biology. I.S. SRL2 spL2/dipL2 is the incumbent of the Pontecorvo Professorial Chair of Cancer Research.

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17366 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806604105 Politi et al.