Article Cite This: Cryst. Growth Des. 2018, 18, 1768−1775
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Noninvasive Microcomputerized X‑ray Tomography Visualization of Mineralization Directed by Sea Urchin- and Nacre-Specific Proteins Martin Pendola and John Spencer Evans* Laboratory for Chemical Physics, Center for Skeletal and Craniofacial Biology, New York University, 345 East 24th Street, New York, New York 10010, United States S Supporting Information *
ABSTRACT: The biomineralization process offers novel principles for crystal engineering and solid-state chemistry, but to achieve this we must first understand how organisms such as the mollusk and sea urchin craft skeletal elements such as the shell and embryonic spicule, respectively. In vitro studies of mollusk- and sea urchin-associated proteins reveal that these proteins form hydrogel particles that control the nucleation process, assemble mineral nanoparticles, and modify the surfaces and interiors of existing crystals. However, visualization of these processes is hampered by destructive and invasive methods such as those used for conventional scanning electron microscopy/transmission electron microscopy. In this report we detail the novel use of microcomputerized X-ray tomography (μCT) imaging to nondestructively investigate the in vitro calcium carbonate mineralization process in the presence of a recombinant sea urchin spicule matrix protein, rSpSM50, and a recombinant mollusk shell nacre protein, rPif97. Relative to the protein-free control scenario, both proteins generate calcite crystals that are consistent with the results obtained from previous studies, but with μCT we discovered new features; each protein generates a different number of mineral deposits, exhibits unique domains or motifs, and creates a stratification of mineral phases into layers. These results coincide with the known function of these proteins in vitro and in situ, and provide new information regarding biomineralization protein hydrogels and how they influence nucleation and crystal growth. We foresee that μCT imaging could, for appropriately sized systems, offer a bridge between in vitro experiments and in situ investigations of time-resolved nucleation and crystal growth phenomena in organisms, tissues, and synthetic materials.
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INTRODUCTION
stabilization/transformation, and nanoparticle assembly. Thus, to better understand the formation of biological composites, we need to understand the role(s) that sea urchin and pearl oyster protein families play in mineralization. Previously, studies have shown that sea urchin spicule matrix21−23 and mollusk shell nacre layer24−26 proteins form ion-responsive “smart” mesoscale hydrogel particles that control the early events in in vitro nonclassical nucleation processes27−29 and organize and assemble nanoscale mineral deposits within their matrices. This is an important event, in that subsequent assembly of mineral-impregnated hydrogel particles would position mineral nanoparticles to eventually form a crystal.30,31 Two protein hydrogelators which have been studied in this regard are the purple sea urchin (Strong-
The formation of calcium carbonate-based skeletal elements by oceanic organisms is a fascinating process that offers new and important insights into composite material formation. Two model organisms that have been extensively studied are the sea urchin1−9 and the mollusk.10−20 In both cases, the mineralized skeletal elements that form follow a developmental plan that involves the creation of a protein-based matrix into which nanoscale mineral deposits are formed and assembled into mesoscale single crystals of fracture-resistant calcite (sea urchin)1−8 or aragonite (mollusk).10−14 This process involves the formation of an amorphous precursor, amorphous calcium carbonate (ACC),2−5,16 which transforms into the appropriate crystalline polymorph (spicule = calcite; shell nacre = aragonite) during skeletal formation. In both organisms, there are protein families or proteomes6−9,17−20 that are expressed during this mineralization process that guide various aspects of single crystal formation, such as nanoparticle formation, ACC © 2018 American Chemical Society
Received: November 29, 2017 Revised: January 6, 2018 Published: January 24, 2018 1768
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Figure 1. Snap-cap polypropylene vials utilized in the microCT study. Note the round region at the center of the vial bottom exterior (arrow); this is a manufacturing “stub” on the outside of the vial that remains from the sprue injection manufacturing process. This stub protrudes approximately 0.5 mm into the interior of the vial as a hemispherical region. Outside dimensions: 12.7 mm diameter, 13.03 mm height; inside dimensions: 7.37 mm diameter, 5.72 mm height. The vials hold approximately 200 μL of liquid.
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ylocentrotus purpuratus) embryonic spicule matrix protein, SpSM50,22 and the Japanese pearl oyster (Pinctada f ucata) framework shell nacre layer protein, Pif97. Both proteins are strong hydrogelators; they form mesoscale protein hydrogel particles that contain mineral deposits.22,24 In addition, both protein hydrogel particles modify the surfaces of growing calcite crystals and form intracrystalline protein inclusions, thereby creating nanoporous crystals.22,24 In previous studies of both proteins, the organization and assembly of hydrogel-mineral particles was analyzed using electron microscopic imaging (scanning electron microscopy (SEM)/transmission electron microscopy (TEM)),22,24 but because of sample dehydration requirements, hydrogel collapse and distortion of mineral particle positioning occurred. Moreover, selective area imaging performed in these studies did not provide an overall picture of what occurs during the in vitro nucleation process. Thus, additional studies using appropriate nondestructive imaging methods and more inclusive sampling are necessary to advance our understanding of protein−directed mineral particle assembly and organization. In this present report we detail the use of microcomputerized X-ray tomography (microCT or μCT) to analyze and quantitate the in vitro calcium carbonate nucleation and crystal growth processes controlled by recombinant variants of SpSM50 (rSpSM50) and Pif97 (rPif97) proteins. Typically, μCT imaging has been used to examine a wide range of biological and material samples,32−38 but to our knowledge this is the first time that μCT has been applied to study and quantitate mineralization in aqueous solution. Using microvials, this noninvasive approach allows surveying of the entire mineralization volume and avoids dehydration distortions associated with past EM studies,22,24 thus preserving the positioning and organization of mineral deposits. We find that, compared to protein-free control scenarios, both rSpSM50 and rPif97 induce the formation of calcite mineral particles that are spatially distributed and exhibit multilayer stratification, with rPif97 generating fewer crystals and rSpSM50 generating the largest number of mineral particles in this study. Further, motif or domain distributions for these mineral particles differ from the control scenario, with rSpSM50 generating more motifs with smaller dimensions and rPif97 promoting mineral particle clustering leading to fewer motifs that are larger in size. In conclusion, we establish that both rSpSM50 and rPif97 hydrogel particles exhibit signature properties of mineral formation, distribution, and particle sizes. These differences may be important for the role that each protein plays in the formation of fracture-resistant biological composites.
EXPERIMENTAL SECTION
Protein Preparation and in Vitro Mineralization Assays. Recombinant S. purpuratus SpSM50 (rSpSM50) and P. fucata Pif97 (rPif97) proteins were subcloned and expressed in Escherichia coli bacteria and purified as described in previous studies.22,24 Purified proteins were dissolved in 30 nm ultrapure Molecular Biology grade water (Fisher Scientific, USA) to create stock solutions for assay use. In vitro biomineralization assays (10 mM CaCl2/NaHCO3/Na2CO3, pH ≈ 8.0, 60 min duration, 25 °C) were performed as described previously,22,24 using a final assay volume of 200 μL and hinged-lid sealable plastic polypropylene lab microvials (outside dimensions: 12.7 mm diameter, 13.03 mm height; inside dimensions: 7.37 mm diameter, 5.72 mm height) (LA Container Store, Yorba Linda, CA, USA) (Figure 1). Prior to performing experiments with the microvials, we obtained phantom images of the vials containing 200 μL of ultrapure 30 nm filtered Molecular Biology grade water (Fisher Scientific, USA), which show typical water interference spots that occur in the upper z-axis region of the vials and not at the bottom of the vials (Figure S1, Supporting Information). Subsequently, three sample scenarios were created: a control scenario (no protein was added) and two protein scenarios using aliquots of rSpSM50 and rPif97 stock solutions (final protein concentrations = 1.5 μM). In parallel assays a 5 mm × 5 mm silicon wafer (Ted Pella, USA) was placed at the bottom of each vial to collect mineral and hydrogel deposits that formed by the 60 min period for subsequent microRaman and SEM analyses. μCT Imaging and Data Processing. At the end of 60 min, each mineralization assay vial was mounted to the stage and scanned using a Bruker MicroCT SkyScan high resolution model 1172G (Bruker Scientific, Kontich, Belgium) with a 59 kV source voltage and 167 μA current. The scanning process involved a round scanning trajectory and step-and-shoot protocol, with each image requiring 0.3 deg/step and 780 ms exposure time. Approximately 1300 images with a final resolution of 4.99 μm per pixel were acquired for each sample over an 80 min period; thus, the total mineralization assay elapsed time was 140 min. For each sample the sequence of images was exported as a series of bmp image files. For every sample we identified the first xy-plane or transverse image showing mineral deposits at the bottom of the vial; then progressing in the +z-axis direction, we selected the next 189 transverse images to complete a sequence of 190 images from the bottom of the vial up to approximately 950 μm in height. Collectively, these images encompassed the mineral deposits in toto generated in each assay. Using PhotoShop CS5 (Adobe Systems, Inc., USA), we selected mineral deposits in each xy-plane frame using the color range selection tool, with a low fuzziness factor to prevent selection of false positive objects. Once the objects were selected, we subtracted all the other elements in the image using the inverse selection tool to retain only mineral deposits. This process was repeated for all 190 images. The 190 images that were produced were then used for threedimensional (3D) reconstruction. These image stacks were imported 1769
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Figure 2. (A) μCT images (along the z-axis) of vials containing mineral deposits formed in control [(−) protein] and rSpSM50, rPif97 mineralization assays. Arrows denote the presence of mineral particle clustering. Scale bars = 1 mm. (B) 3D reconstruction of the same samples. The raised hemispherical region that represents the production spar can be clearly seen internally at the bottom of each vial. Orientation xyz axes shown.
Figure 3. (A) Total mineral particle number and (B) average mineral particle area analyses for the three mineralization scenarios. For (B), one-way ANOVA (analysis of variance) shows that the differences between samples is significant (p > 0.0001). kV and a probe current of 100 pA.22,24 Prior to analysis, samples were coated with iridium (3 nm layer) using a Cressington 208HR sputter coater with thickness controller.22,24
into ImageJ 1.46r 64bit (NIH, USA) and rendered into 3D using the volume viewer tool with tricubic (sharp) interpolation. From this 3D reconstruction, we selected two transverse images with a 0.25% (low magnification) and 50% (high magnification) scales. For further quantitative analyses of mineral deposits, we used the volume viewer reconstruction and analyze commands in ImageJ, and counted the number of mineral particles and measured their dimensions in each sample. In addition, the volume viewer images produced with ImageJ were analyzed using MountainsMap 7 (Digital Surf, France). Using the tool Binary segmentation, the events present in each image were grouped based on the shapes’ respect to the background, and represented as a single motif, with different colors assigned for each motif. MicroRaman and SEM Analyses of Mineral Deposits. Using the deposits which collected onto Si wafers at the bottom of each assay vial, all samples were washed 3 times with calcium carbonate− saturated methanol and dried overnight.22,24 Subsequently, these Si wafers were analyzed using micro-Raman spectroscopy (Thermo Scientific DXR Raman microscope in dark field mode, 532 nm laser wavelength, 6 mW power, 1800/900 grating, 25 μM pinhole aperture, 16 exposures per sample at MP50× magnification). The spectra were processed using the OMNIC Atlus software designed for the instrument.22,24 Other parallel wafers were imaged using a Merlin (Carl Zeiss) field emission SEM (FESEM) with an Everhart-Thornley type secondary electron detector (SE2) at an accelerating voltage of 2
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RESULTS The purpose of this μCT imaging study is to noninvasively detect and analyze X-ray attenuating calcium carbonate mineral particles that form in the presence and absence of the two hydrogelator proteins within a polypropylene microvial system. The mineralization assay used in this study is tuned to stable calcite production.22−26 Our goal is to distinguish the quantity, size, distribution, and organization of mineral particles relative to each other and to a protein-free scenario. The hydrogels themselves possess scarce X-ray attenuation features and thus due to low imaging contrast will not be directly detected by μCT. However, the influence of the hydrogels on mineral formation (dimensions, location, distribution) will be indirectly detected, as shown below. The use of sealable polypropylene plastic vials minimizes evaporation and carbon dioxide outgassing from carbonate/ bicarbonate species39 during the assay and subsequent imaging period and minimizes the backscattering X-ray beam artifacts 1770
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Figure 4. μCT XZ-sagittal plane volumetric sections (4 mm × 4 mm × 1.9 mm) of microvial mineralization samples. In all samples, note the presence of suspended mineral particles above the bottom of the vials. In the case of the protein samples, note the presence of two layers of mineral particles (labeled as 1 and 2). Note also the presence of a large, suspended mineral cluster in the rPif97 image (white arrow). Scale bar = 1 mm; height of each image = 1.9 mm.
(Figure S1, Supporting Information) associated with glass containers. Moreover, given the dimensions of the vials, we can completely image and quantitate the entire mineralization assay volume, something not possible in previous studies.22−26 Spicule and Nacre-Associated Hydrogel Particles Differ in Mineral Nucleation. In Figure 2, we present the z-axis projection image (Figure 2A) and 3D reconstruction (Figure 2B) images of the liquid volumes contained within each sample vial with the surrounding plastic vial surfaces omitted. Two features are noted in this series of images. (1) Both images reveal the total population of mineral particles generated by each sample. We tabulated the total number of particles detected in each sample (Figure 3A) and the average area occupied by all the particles in each sample (Figure 3B). From these tabulations we find that the number of detected crystals and occupied area follows the relationship: rSpSM50 > control > rPif97. Both proteins are structurally similar and are nearly equal in aggregation propensity,22,24 and thus these factors probably do not contribute significantly to these proteinspecific results. However, it is known that Pif97 inhibits calcite crystal growth,18,24 and thus the lower number of mineral particles formed in the presence of rPif97 is logical. Likewise, rSpSM50, which forms a transient vateritic ACC phase in vitro that quickly converts to a more stable calcitic ACC phase, is expected to promote calcite formation.1,4,5,22 Second, we detected evidence of mineral particle clustering, i.e., the presence of particles which appear to be either in contact or closely spaced with one another (Figure 2A, arrows). We observed that the occurrence of clustering follows the relationship: rPif97 > control > rSpSM50, i.e., the inverse of the particle number relationship. In the case of the control, the
presence of clustering may be attributable to epitaxial growth. However, for rPif97, given the millimeter dimensions of the formed clusters, it is unlikely that epitaxial growth contributed to cluster formation. More likely, the long-range clustering that we observe in the presence of rPif97 reflects protein hydrogel adhesion to mineral particles that leads to clustering during mineralization.24 From the 3D reconstruction and z-axis transverse images, we conclude that relative to the control scenario rSpSM50 generates a larger number of mineral particles, whereas rPif97 generates fewer mineral particles (i.e., acts as a calcite inhibitor) over the same time interval but promotes particle clustering. Sagittal Imaging Plane Reveals Mineralized Stratification. The 3D reconstruction images in Figure 2B suggest that in addition to mineral precipitation at the bottom of each vial, there exist mineral particles which are suspended in the assay liquid above the vial bottoms. To assess this, we examined cross-sectional xz-plane sagittal images taken at the midline of each vial (Figure 4; Figure S2, Supporting Information). In all samples we note the presence of mineral particles that are suspended in the liquid above the vial bottoms. Presumably, these are mineral particles whose masses or densities are lower than those which reside at the bottom of the vials. Thus, these particles were unable to settle out during the assay and imaging time periods. Of the three samples, it appears that rPif97 possesses a larger number of suspended mineralized particles compared to either the control or rSpSM50 samples, and features evidence of cluster formation (white arrow, Figure 4). This is consistent with rPif97 hydrogel particles creating large micron-sized protein inclusions within calcite during these assays,24 which would make these crystals more nanoporous 1771
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Figure 5. (A) Enlargement of representative μCT images of mineral particles that deposited in (−) protein, rSpSM50, and rPif97 mineralization assays. Scale bars = 4 μm. (B) Corresponding representative SEM images of mineral deposits collected from parallel mineralization assays. Note the close correspondence in morphologies between microCT and SEM images of crystals in each sample, and the presence of protein aggregates or hydrogel phases that have deposited onto the Si wafers or crystals (arrows). In SEM images scale bars correspond to either 2 or 1 μm.
particles that form in the presence of both proteins are >5 μm,22,24 we were able to detect both individual and clustered mineral particles formed in all assays. Obviously, aliasing becomes a problem at higher magnification, and thus we also performed SEM imaging on mineral deposits collected on Si wafers in parallel assays to determine crystal morphologies more precisely (Figure 5B). A comparison of the μCT and SEM images for the control and rSpSM50 scenarios reveals overall agreement: we observe the typical rhombohedral morphologies associated with calcite in these samples (Figure 5A,B). Moreover, as shown by the arrows in the SEM images, we also confirm the formation of protein phases or hydrogels depositing onto the Si wafers or onto the crystals themselves (Figure 5B). Using microRaman spectroscopy, the calcite polymorph was also confirmed for the modified mineral crystals generated by rPif97 samples (Figure S3, Table S1, Supporting Information). Note that we cannot confirm nor rule out the presence of ACC deposits in these assays. Hypothetically, any stabilized ACC that formed in the assay may be suspended in solution and probably associated with the protein hydrogel phase (Figure 4). We also note that for each protein hydrogel, signature crystal morphologies were observed that correlate with previous assay results (Figure 5A,B): The rounded, highly modified calcite crystals of rPif9724 are easily distinguishable from the smaller, rhombohedral crystals of rSpSM5022 using μCT. However, as one might expect, the nanotextured surface modifications created by rSpSM50 that are readily detected by SEM22 cannot be detected by μCT. Thus, the microvial assays qualitatively replicate the results obtained in past in vitro mineralization studies for each protein hydrogel,22,24 and μCT imaging affirms that each protein differentially regulates calcite crystal size and morphology, but cannot provide information regarding protein-induced surface or subsurface modifications of crystals. Protein Hydrogel-Mediated Organization of Mineral Particles. We now examine the arrangement or organization of
and potentially more buoyant in aqueous solution. But perhaps the most intriguing feature noted in these sagittal images is that both the rPif97 and rSpSM50 samples form stratified mineralized phases that settle out over time (denoted as Layers 1 and 2) and are distinguishable by their degree of X-ray attenuation. In comparison, the control sample possesses only a single layer, and thus the stratification effect is protein-driven. In the case of rPif97, Layer 1 appears to possess less X-ray attenuation compared to Layer 2, and interestingly, the reverse is true for the rSpSM50 sample. Note that the dimensions of the rSpSM50 layers are greater than those generated by either rPif97 or the control scenario, and that the rSpSM50 Layer 2 is significantly thicker and less dense than Layer 1. We believe that this stratification phenomenon originates from one or more of the following phenomena: (1) Protein differences in mineral particle organization, crystallinity (i.e., calcite crystals versus stabilized ACC particles), size, and assembly22,24 induced by the hydrogel phases that lead to stratification differences; (2) differences in protein content associated with the mineral particles, either through external surface associations or internal inclusions (i.e., intracrystalline protein-containing nanoporosities),2,3,14,15,22,24 which would alter the buoyant densities, sedimentation, and spatial positioning of the mineral phases, leading to the protein-specific stratification effects. In conclusion, μCT imaging reveals a new in vitro protein-driven biomineralization phenomenon: nacre and sea urchin matrix proteins generate a mineral stratification effect that is proteinspecific. We believe that this layering phenomenon reflects the influence of protein hydrogel particles on the calcium carbonate nucleation, transformation, stabilization, assembly, and crystal growth processes.22,24 Analyses of Mineral Particles and Hydrogel Phases Generated by rSpSM50 and rPif97. Using image processing we visualized the mineral particles that were generated in the control, rSpSM50, and rPif97 assays (Figure 5A). Given that μCT resolution is approximately 5 μm, and that most mineral 1772
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Figure 6. Domain mapping of microvials along the z-axis. Red is considered as empty or low density areas where mineral deposits have no relevant proximity to each other; other colors represent distinct domain types (colors were arbitrarily assigned). Each axis represents 1.7 cm in length. The number of mineral particle-containing domains is provided below each image.
matrix protein, rSpSM50.22 Both proteins are strong hydrogelators associated with mesoscale crystal formation from the assembly of mineral nanoparticle precursors,1−5,10−14,16 and thus make excellent model systems for this type of study. We find that each protein has signature mineral particle formation and organizational characteristics. Specifically, rSpSM50, which oversees the transformation of efficient space-filling ACC nanoparticles40 to single crystal calcite in the developing spicule,1,4,5 generates the largest number of crystals with the smallest dimensions that cover the largest area of the vials (Figures 2 and 3), produces the largest number of discrete crystal domains (Figure 6), and creates stratified layers of mineralized phases in the assay (Figure 4). In contrast, rPif97, a known calcite inhibitor,18,24 creates fewer mineral particles (Figures 2 and 3), but fosters larger mineral domains (Figure 6) with evidence of crystal clustering (Figures 2 and 4). This protein also creates stratified mineralized phase layers (Figure 4), but, compared to rSpSM50, this stratification effect is reduced in dimension, and X-ray attenuation is not as pronounced compared to rSpSM50 (Figure 4). Thus, μCT imaging has detected new phenomena regarding rPif97- and rSpSM50-directed in vitro calcium carbonate nucleation and crystal growth that were previously undetected using invasive or destructive imaging methods.22,24 We believe that the in vitro mineralization behavior of rPif97 and rSpSM50 protein hydrogels provides new insights vis a vis the involvement of native Pif97 and SpSM50 in the mineralization processes that occur within the mollusk shell nacre layer and the sea urchin spicule, respectively. It is known that calcium carbonates such as calcite form from ACC nanoparticle assembly in vitro,30,31,41 and given that both skeletal elements arise from nanoparticle formation and assembly it is interesting to note that both proteins generate mineral particles of a specific quantity, size, and area (Figures 2 and 3). Given that SpSM50 is the most abundant matrix protein in the embryonic spicule,6−9 it may be the responsibility of this protein hydrogel to promote nucleation and limit the size of the mineral deposits (Figures 2−4) to facilitate spacefilling arrangements40 and promote the assembly of single crystal calcite. Conversely, it is known that Pif97 acts as a calcite inhibitor and exists as a complex with Pif80 and n16.3 in the nacre layer of the oyster shell,17,18 and thus the formation of
mineral particles which form during the mineralization assay period. This can be done using binary segmentation analysis (MountainsMap software package), where mineral particles are selected by shape and then groups, “domains”, or “motifs” are defined and limited by the highest percentage of mineral particles that cover the smallest possible surface area. Domain boundaries are denoted by different colors, with red assigned to areas of lowest particle density or where no crystals exist (Figure 6). Using the control scenario as a reference (2969 domains) we can observe that rSpSM50-associated mineral particles occupy smaller but more numerous domains (4966, nearly 2× the number of the control scenario). Note that smaller mineral particle sizes have been reported to be efficient in space-filling arrangements within sea urchin skeletal elements,40 so the smaller, numerous domains generated by rSpSM50 are qualitatively consistent with these in situ findings. Conversely, the mineral particles generated in the presence of rPif97 exist in fewer domains (2264) than either the control or rSpSM50 samples but clearly possess larger domain regions and feature significant open regions where little or no mineral particles occur (Figure 6). We note that the domain phenomena (Figure 6) correlates with clustering phenomena, i.e., rPif97 > control > rSpSM50 (Figure 2). We presume that the protein hydrogel particles influence the formation of mineral particle domains in terms of number and size, and this may have relevance for the formation of the crystalline phase from amorphous mineral nanoparticle precursors in both the nacre layer and the spicule matrix.
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DISCUSSION μCT has been applied to the study of many different biological and material samples over the last 10 years and has established itself as a valuable visualization tool for solid and liquid samples.32−38 In our present study, we adapted μCT for a unique purpose: a noninvasive study of real-time protein-driven nucleation of calcium carbonate mineral particles in an aqueous environment. As we have discussed above, this approach allowed us to completely image the entire mineralization assay volume. In doing so, the method yielded some very interesting information regarding the in vitro nucleation and crystal growth functionality of a recombinant mollusk shell nacre protein, rPif97,24 and a recombinant embryonic sea urchin spicule 1773
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fewer calcite crystals, induction of crystal clustering, and creation of larger mineral domains may reflect Pif97 participation within this ternary protein complex. The other interesting feature is the stratification of mineral particles in each protein assay (Figure 4). This layering phenomenon suggests that the protein-mediated formation of mineral phases over time is not a homogeneous process, but a heterogeneous one that produces different particle dimensions and densities that reflect protein hydrogel content and/or association with the mineral phase. At this time we do not understand the significance of this in vitro phenomenon and its relevance to in situ mineralization events, and thus additional research into this process will be required in order to establish its importance. Finally, the clear strength of the μCT approach is that the systems under study remain nearly undisturbed and the instrumentation permits the noninvasive study and quantitation of nucleation and crystal growth. Potentially μCT could allow for the comparisons of in situ nucleation processes alongside parallel in vitro processes in a wide range of synthetic systems, materials, or organisms and tissues whose dimensions are compatible with μCT. This approach can also allow timeresolved monitoring or kinetics of nucleation processes, albeit at lower resolution compared to EM methods. This is a particularly attractive and powerful “bridging” scenario for linking crystal growth events that occur in vitro and in situ as a function of time, whether directed by proteins or other additives such as polymers. Finally, using appropriate contrasting agents,34,37 it may be possible to visualize the organic or protein-based hydrogel within in vitro and in situ environments, thereby gaining additional insight into hydrogeldirected mineral particle formation, assembly, and organization. We intend to pursue these avenues of research in future applications.
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ACKNOWLEDGMENTS
This report represents contribution number 90 from the Laboratory for Chemical Physics, New York University.
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ABBREVIATIONS rSpSM50 = recombinant version of the Strongylocentrotus purpuratus purple sea urchin spicule matrix protein 50 rPif97 = recombinant version of the Pinctada f ucata Japanese pearl oyster shell framework nacre protein Pif97 μCT = microcomputerized X-ray tomography REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.7b01668. 3D and XY-transverse images of microvials containing 200 μL of 30 nm filtered molecular biology grade water, 25 °C (water phantom) (Figure S1); Sagittal XZ plane images obtained from control, rPif97, and rSpSM50 mineralization assays (Figure S2); microRaman spectra of rPif97 mineralization assay mineral deposits (Figure S3), and Raman band assignments for calcium carbonates (Table S1) (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Address: Laboratory for Chemical Physics, Division of Basic Sciences and Center for Skeletal and Craniofacial Biology, New York University College of Dentistry, 345 E. 24th Street, New York, NY, 10010. Tel.: (212) 998-9605; Fax: (212) 995-4087. E-mail:
[email protected]. ORCID
John Spencer Evans: 0000-0002-9565-7296 Funding
This research was supported by the Life Sciences Division, U.S. Army Research Office, under Award W911NF-16-1-0262. Notes
The authors declare no competing financial interest. 1774
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Crystal Growth & Design
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DOI: 10.1021/acs.cgd.7b01668 Cryst. Growth Des. 2018, 18, 1768−1775
