Communication pubs.acs.org/crystal
Focused Ion Beam Tomography Reveals the Presence of Micro‑, Meso‑, and Macroporous Intracrystalline Regions Introduced into Calcite Crystals by the Gastropod Nacre Protein AP7 Eric P. Chang, Gabrielle Williamson, and John Spencer Evans* Laboratory for Chemical Physics, Center for Skeletal Biology, New York University College of Dentistry, 345 Easts 24th Street, New York, New York 10010, United States S Supporting Information *
ABSTRACT: Intracrystalline modification of calcium carbonates by macromolecules is a fascinating process that offers insights into potential pathways for modifying the material properties of inorganic solids. Recently, we reported on the induction of intracrystalline nanoporosities within calcite by the nacre layer intracrystalline protein, AP7 (Haliotis rufescens). In this report we revisited this AP7-mediated phenomenon and tracked time-dependent intracrystalline porosity formation during in vitro mineralization using FIB/SEM serial milling. We find that AP7 induces intracrystalline nanoporosities as early as 1 min of elapsed assay time. Quantitation of pore regions confirms that average cross-sectional volume (ACSV), average void volume (AVV), and percent porosity parameters increase over time, leading to the formation of porous calcite crystals with a high surface-to-volume ratio. FIB serial milling, SEM imaging, and 3-D tomography revealed the presence of unexpected semicontinuous channels and cavities in the subsurface regions of a representative 60 min assay crystal. The random locations of these intracrystalline features are limited to the top and sides of the calcite crystal, which correspond to the sites of AP7 protein phase deposition during mineral formation. This random porosity distribution was also documented for protein-containing voids within nacre aragonite tablets in situ. In some instances we observed geometric relationships between adjacent channels and cavities. Interestingly, all three IUPAC-defined material porosity categories (micro-, meso-, and macro-) were represented in the AP7-treated crystals. Thus, the deposition of AP7 protein phases onto calcite surfaces induces surface nanoparticle nucleation and subsurface multiscale intracrystalline porosities and interconnected channels.
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poorly understood, and thus our ability to develop novel organic-occluded inorganic materials is limited. Recently, an interesting in vitro phenomenon was reported for the intrinsically disordered intracrystalline nacre protein AP7 (66 AA, 7565 Da) of the Pacific red abalone gastropod (Haliotis rufescens).5,7,8 AP7 is occluded within aragonite tablets5 of the nacre layer and in the absence of Mg2+ forms protein phases that promote calcite nanoparticle formation on the surfaces of existing calcite crystals in vitro.7 These nanoparticles are highly oriented, periodically separated by spaces or gap regions at the surface (Figure 1), and resemble the arrangement of mineral structural units found within biogenic single crystal calcite and aragonite that contain intracrystalline macromolecules.10 However, what is most surprising is that intracrystalline porosities appear over time within these AP7-modified calcite crystals.7 These porosities are far more numerous than those found within protein-deficient control crystals,7 appear random in their subsurface location and dimension, and resemble occluded regions produced by macromolecular additives12−14 and by proteins.1,3 A somewhat similar in vitro calcite porosity phenomenon was also generated
iomineralization proteins are known to be effective regulators of calcium carbonate mineral nucleation and crystal growth, and in some cases, these proteins are capable of subsurface modifications to the mineral phase.1−12 In particular, there are a number of proteins or other biomacromolecules which become occluded within calcium carbonate crystals, typically at low weight percent.1−6 This incorporation affects the texture, lattice strain, and anisotropy of the crystal and promotes fracture toughness.1−4,10−12 It is believed that this intracrystalline incorporation process initiates at the amorphous calcium carbonate (ACC) mineral precursor stage that precedes crystalline transformation.13−17 Additives, such as proteins, associate with or stabilize ACC and become incorporated into the mineral phase during the amorphous-to-crystalline transformation process.10−16 To monitor this process a number of in vitro mineralization systems have been developed wherein additives such as amino acids,15,16 agarose gels,14 recombinant nacre proteins,6 and diblock copolymer micelles13 have been successfully incorporated into calcium carbonates. These experiments confirm that a wide variety of molecular and macromolecular organic species can be incorporated within crystals with subsequent modifications of the physical properties of these crystals.6,13−16 However, the phenomenon of intracrystalline modifications by biomineralization proteins is still © 2015 American Chemical Society
Received: February 13, 2015 Revised: March 16, 2015 Published: March 18, 2015 1577
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Figure 1. (A) SEM image of a calcite crystal formed in AP7-containing assays (1 h duration). (B) Higher magnification image of (A), denoting orthogonally ordered nanoparticle surface features that include spaces and gaps. previous mineralization studies.7,8 The final pH of the reaction mixture was measured and found to be approximately 8.0−8.2.7−9 Mineral and protein deposits formed during the assay were captured on 5 × 5 mm2 Si wafer chips (Ted Pella, Inc.) that were placed at the bottom of the vials. Upon completion of a mineralization assay, the Si wafers were rinsed thoroughly 3× with calcium carbonate saturated methanol and dried overnight prior to analysis.7−9 Standard imaging of the Si wafers extracted from the mineralization assays was performed using a Merlin (Carl Zeiss) field emission SEM (FESEM) using either an Everhart-Thornley type secondary electron detector (SE2) or an annular secondary electron detector (in lens) at an accelerating voltage of 2.0 kV and a probe current of 600 pA.7 Prior to analysis, samples were coated with iridium using a Cressington 208HR sputter coater with thickness controller attachment. Imaging of internal crystal morphology was performed using a Zeiss Auriga Small Dual-Beam FIB-SEM.21−25 For these analyses all samples were first coated with 4 nm iridium prior to SEM imaging, then coated with 50 nm of Au prior to performing FIB. A 30 kV 120 pA gallium ion beam was oriented perpendicular to the sample by tilting the sample stage to 54° and utilized to mill 15 nm serial cross sections. SEM images of cross-sectioned surfaces were then obtained using a 2.0 kV 600 pA electron beam and a secondary electron detector at a working distance of 5.0 mm. Images of surfaces containing electron beam damage were created for comparison to images of undamaged surfaces but were not used for the purposes of discussion in this publication. Images were taken shortly after cross sectioning to limit the exposure of the uncoated surfaces to the electron beam.21−25 For each time point sample, 10 serial cross-section SEM images were taken from 3 to 4 representative crystals and analyzed to compute the volume of the crystal cross-section and the volume of the void regions. These values were quantitatively estimated from the length of each pixel and the thickness of each cross-section (nm units), and the average cross-sectional volume (ACSV, nm3) and average void volume (AVV, nm3) were calculated from these values. The percent porosity was then derived from ratio of AVV to ACSV × 100. For the 60 min crystal, FIB SEM serial sectioning techniques (200 cross sections) and stack processing21−25 were used to reconstruct a 3-D model using Avizo Fire 6.2 software. (FEI, Inc., Hillsboro, OR, USA). The number of pixels in each SEM void region was measured using ImageJ and only porous regions conserved between two consecutive cross sections were measured (Supporting Information, Figure S1).
by Pinctada fucata n16.3 framework nacre protein phases.9 This AP7-directed porosity process potentially offers insights into how proteins generate intracrystalline features in crystals in vitro9 and in vivo,1−12 and offers an alternative route to the creation of porous inorganic solids without the need for a template.18−21 Unfortunately, our understanding of this intracrystalline porosity formation process is incomplete, owing to the fact that these initial studies only examined a limited number of focused ion beam (FIB)-generated crosssection images at one assay time point.7,9 As a result, these studies were unable to address (1) the time-dependent formation of these voids or pores and (2) the threedimensional location, dimension, and distribution of these pores within the subsurface regions of nucleating calcite crystals. For these reasons we revisited the phenomenon of AP7 protein-induced crystal porosities by tracking their timedependent formation during in vitro mineralization assays and assessing pore sizes and distributions within representative calcite crystals using FIB/SEM serial milling techniques.21−25 We find that the formation of subsurface porosities commences as early as 1 min and continues to develop over time as the overgrowth mineral phase increases in dimension. Using FIB/ SEM tomography21−25 on a representative mature AP7modified crystal (60 min), we mapped porosity locations and discovered a high degree of complexity and interconnectivity as evidenced by the presence of interconnected chambers or channels within the subsurface mineral phase. Further, the dimensions of these porous regions span the broad classification scheme for material porosities established by IUPAC:26 there are micro- (2 nm, < 50 nm), and macroporous (>50 nm) regions within the modified crystals. Thus, the AP7 protein phase simultaneously changes both the external and internal physical nature of calcium carbonate crystals over time, and this process ultimately leads to the creation of multiscale intracrystalline porosities.1,3
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EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION In previous studies, FIB/SEM milling revealed the presence of multiple intracrystalline porosities within nucleating calcite crystals that were repetitively coated by AP7 protein phases over a 60 min period. These subsurface features were found to be almost nonexistent in protein-deficient control crystals (Supporting Information, Figure S1).7 We now extend these FIB/SEM subsurface observations to include samples obtained
Stock concentrations of chemically synthesized AP7 were prepared using unbuffered deionized distilled water and lyophilized protein.7,8 Mineralization assays followed published protocols7−9 and were conducted by mixing equal volumes of 20 mM CaCl2·2H2O (pH 5.5) and 20 mM NaHCO3/Na2CO3 buffer (pH 9.75) to a final volume of 500 μL in sealed polypropylene tubes and incubating at room temperature for 1, 5, 15, and 60 min. An aliquot of AP7 stock solution was added to the calcium solution prior to the beginning of the assay, with final protein concentration = 100 μM, identical to that utilized in 1578
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from earlier stages of this protein-mediated process (i.e., 1, 5, 15 min, Figure 2). Compared to protein-deficient control
Figure 3. Time-dependent subsurface parameters for AP7 protein phase-modified calcite crystal void/pores: average cross-sectional volume (ACSV), % porosity, and average void volume (AVV). These terms are defined in the Experimental Procedures and were determined from 10 FIB-SEM serial cross sections per crystal at each time point.
Figure 2. SEM images of FIB-sectioned calcite crystals. (−) AP7 represents the typical protein-deficient assay calcite crystal (60 min assay time), which features minimal porosities (arrows). Contrast this finding to FIB sectioned crystals obtained from AP7-containing mineralization assays of 1, 5, 15, and 60 min duration, where one can clearly denote multiple subsurface void or pore regions (white arrows). For clarity, expanded SEM images for time points 1, 5, 15, and 60 min can be found in Supporting Information, Figure S2−S5.
formation of intracrystalline porosities is prevalent during the later stages (>15 min) of in vitro crystal growth. Given that the greatest degree of intracrystalline porosity is observed at 60 min, we performed FIB tomography of a representative AP7-modified calcite crystal obtained from this time point. This involved repetitive serial FIB sectioning and SEM imaging as described in Experimental Procedures. From these serial images we generated both a surface and an internal 3-D reconstruction of this crystal (Figure 4). This tomographic image provides us with unique insights into the extent of subsurface calcite crystal modifications induced by AP7. The first notable feature is that the intracrystalline porosities are localized within the top and sides of the growing crystal. Interestingly, these interior regions correlate with surface locations where AP7 protein phase deposition occurred during the early phases of crystal growth (i.e., 1 min, Figure 5).7 Note that the bottom surface of the crystal is porosity-deficient (Figure 4), presumably because this region was in contact with the Si wafer and thus inaccessible to depositing protein phases. The second notable feature is that the locations, dimensions, and morphologies of the intracrystalline voids are random (Figure 4, images A2, C2, E2), which reflects the amorphous nature and dimensional heterogeneity of the randomly deposited protein phases (Figure 5).7 The random patterning and morphology of the AP7-generated porosities are reminiscent of the “footprint” created by the recombinant intracrystalline protein, perlucin, when it becomes incorporated within in vitro calcite crystals,6 and to some extent mirror the
scenarios, crystals that form in the presence of depositing AP7 protein phases clearly exhibit intracrystalline nanoporosities as early as 1 min of elapsed assay time (Figure 2). As time progresses, the crystals increase in overall dimension and we continue to observe porosity formation as surface nucleation events progress in the presence of AP7 protein phases. At 60 min, the crystals feature extensive porosity distribution throughout the outer mineral layer. Thus, the formation of intracrystalline nanoporosities coincides with the reported initial deposition of AP7 protein phases onto exposed calcite crystal surfaces7 at 1 min and later within the in vitro assays. We next quantitated pore regions using 10 FIB/SEM serial cross-section images obtained from 3 to 4 representative crystals at each of the 4 time points (Supporting Information, Figures S2−S5). As shown in Figure 3 and described in the Experimental Procedures, we calculated the average crosssectional volume (ACSV), average void volume (AVV), and percent porosity as a function of assay time. The results are quite intriguing: (a) ACSV values increase by an order of magnitude from 1 to 5 min and peak at 15 min; (b) AVV values increase by a factor of 3 from 5 to 15 min and again from 15 to 60 min; (c) percent porosity remains relatively constant from 1 to 15 min, then increases by a factor of 5 at 60 min. Hence, the 1579
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Figure 4. FIB/SEM tomography of a representative AP7-modified calcite crystal (60 min assay time) using FIB SEM images obtained from 200 serial cross sections. Each perspective A−F features an orientational x,y,z axis and consists of a pair of images [1 = surface topology in green overlaid onto intracrystalline voids/porosities; 2 = intracrystalline voids/porosities in purple]. Note that the intracrystalline porosities exist as single entities and as interconnected channels, and there is evidence of pore alignment (A2, C2, E2) and angular relationships between pores (E2, F2). Scale bars = 2 μm.
single voids and interconnected channels and cavities indicate that the AP7-modified calcite crystal has a high surface-tovolume ratio compared to crystals grown in the absence of AP7 (Figure 2). Intriguingly, the intracrystalline porosity dimensions observed in Figure 4 span the entire range of IUPAC-defined material porosities: micro (2 nm, < 50 nm), and macro (>50 nm) scales (Figure 4).26 Thus, the intracrystalline porosities of AP7-modified calcite are more varied, complex, and interconnected than previously realized.7 In summary, this Communication reports on the intracrystalline nanoporosities and semicontinuous channels introduced within calcite crystals by the intracrystalline nacre protein, AP7.7 These intracrystalline features are randomly distributed, similar to the in situ occurrence of protein-containing voids within mollusk shell nacre tablets.14 Kinetically, we observe the time-dependent formation of intracrystalline porosities (Figures 2, 3) with the most prominent occurrence observed at later stages of in vitro crystal growth (Figure 3). We believe that this is due to changes in bulk crystal size: as calcite crystal dimensions increase over time (Figure 2), additional crystal surface area is available for capturing additional protein phases, which, in turn, induce further surface texturing (i.e., the occurrence of gaps and spaces between nanoparticles, Figure 1) and intracrystalline porosity formation (Figures 2, 3). Note that the in vitro formation of intracrystalline porosities is not unique to AP7 alone: similar features were also detected in calcite crystals that were modified by P. fucata nacre framework n16.3 protein phases.9 What is common for both n16.3 and AP7 is that intracrystalline porosity locations correlate with the gravitydependent deposition of protein phases onto crystal surfaces.7,9 However, the intracrystalline porosities generated by n16.39 were not as extensive in number or location as those generated by AP7.7 This may be attributed to the fact that, compared to AP7, n16.3 promotes directional growth and minimal surface texturing,9 whereas AP7 promotes nanoparticle nucleation and substantial surface texturing (Figure 1).7 Thus, the greater the
Figure 5. SEM image of AP7 protein phase deposition on calcite crystals at 1 min. Arrows denote spreading or migration of the protein phase on the top and sides of the nucleating crystal. Note extensive protein phase deposition on the Si wafer background.
random distribution of protein-containing voids that exist in situ within the aragonite tablets of certain mollusk shells.1,3 Perhaps the most surprising feature is the presence of semicontinuous intracrystalline channels and cavities within the top and side regions of the crystal (Figure 4). These features were not detected in our initial FIB/SEM studies since limited serial sectioning was performed.7 The randomness and curves of these channels are similar to those reported in FIB/STEM tomography studies of agarose hydrogels occluded in calcite.14 Some of the intracrystalline channels or chambers are linearly aligned relative to one another (Figure 4, images A2, C2, E2) or feature angular relationships (Figure 4, images E2, F2). We believe that these geometric features reflect either the original nanoparticle organization that AP7 induced on the surface of these crystals (Figure 1) or the angularity of the bulk crystal calcite lattice (α = β = 90°; γ = 120°) that guided mineral overgrowth in the presence of the deposited protein phase.7 Coupled with our quantitation data (Figure 3), the presence of 1580
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hypothesize that AP7 protein phases create nanopatterned calcite surfaces and mass competition conditions which facilitate protein gel phase incorporation into the overgrowth mineral phase over time. It will be interesting to learn if this is a general phenomenon for protein hydrogel-mediated calcium carbonate crystal growth in Nature.
extent of surface texturing, the greater the entrapment of protein phases at the crystal surface.7 We conclude that the number and extent of intracrystalline porosities are proteindependent and may be controlled by differences in primary sequence features and the effect that each protein phase has on crystal growth.7−9 In previous studies, X-ray microanalyses of protein-associated elemental N and S were performed on voids exposed by FIB/ SEM cross-sectioning in AP7-generated crystals, yet no detectable traces of protein phases were found.7 We believe that there are several plausible explanations for these negative results: (a) Since the N and S energy signals are weak, it is plausible that some protein phase material was retained or entrapped within the nanopores but is below the threshold of detection. (b) Sensitive “soft” protein phase material was damaged and removed from the intracrystalline cavities by the gallium ion beam during repetitive FIB milling.22−25 (c) Protein material was expelled or displaced from cavities by water fluxes or hydrodynamic pressure arising from ACC-to-crystalline transformation during the overgrowth crystallization period.27,28 Although we do not know which explanation is the correct one, we intend to investigate these issues in subsequent studies using cryo-FIB/SEM, nano-SIMS, and other techniques. The FIB/SEM sectioning and imaging techniques21−25 described in this Communication can be considered a semiquantitative approach for visualizing porosities and other features within crystals. In contrast, the FIB/STEM imaging technique utilized by Li et al.14 relies on the use of the FIB beam to “extract” a several hundred nanometer-sized cross section from a given crystal, which is then transferred to a TEM grid for STEM analysis. This technically demanding approach yields high quality data for a limited section of a given crystal. In contrast, the FIB/SEM technique21−25 is less technically demanding yet allows for the visualization (Figures 2, 4) and analyses (Figure 3) of intracrystalline nanoporosities within hundreds of micron-sized crystal cross sections without the need for section extraction and TEM grid transfer. In essence, the two methods complement one another: the FIB/SEM approach has the capability to reconstruct internal porosities within an entire calcite crystal at low resolution (Figure 4), whereas the methods reported by Li et al.14 can provide a highresolution model of crystal cross section. Several in vitro studies have explored the phenomena of intracrystalline inclusions and macromolecular incorporation in calcium carbonates.12,13,18−21,29−31 Some of these studies involved the use of sponge-like templates19,29,30 and hydrogels,12,14,31 both of which create nanoporous calcite. The mechanism for intracrystalline inclusion of templates is straightforward: porosities form as the calcite overgrowth phase expands over and into the accessible porous regions of the template.19,29,30 However, for hydrogels the porosity formation and macromolecular incorporation scenarios are much more complex,12,14 and several mechanisms for these phenomena have been postulated, including force competition, mass competition, hydrodynamic force, and gel resistance to crystallization pressure.31 Given that the AP7 protein phases exhibit gel-like properties,7,8 we postulate that one or more of these hydrogel-based mechanisms are at work in the formation of intracrystalline porosities (Figures 2−4). In fact, the mass competition phenomenon, which facilitates gel incorporation at faster mineralization rates,31 is commensurate with the reported ability of the AP7 N-terminal sequence region to accelerate step growth velocities on calcite hillock surfaces.26 Thus, we
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ASSOCIATED CONTENT
S Supporting Information *
High magnification SEM images of FIB sectioned calcite crystals [protein-deficient control, (+) AP7 at 1, 5, 15, 60 min, Figures S1−S5]. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel.: (212) 998-9605. Fax: (212) 995-4087. E-mail: jse1@nyu. edu. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding
This research was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering under Award DE-FG02−03ER46099. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This paper represents Contribution Number 78 from the Laboratory for Chemical Physics, New York University. ABBREVIATIONS AP7, Aragonite protein 7, Haliotis rufescens; n16.3, n16 aragonite protein, Pinctada fucata, isoform 3; FIB, focused ion beam; ACSV, average cross-sectional volume; AVV, average void volume
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REFERENCES
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