Using HAADF-STEM for atomic-scale evaluation of incorporation of antibacterial Ag atoms in a β-tricalcium phosphate structure

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Introduction
Calcium phosphates (CaPs) have long received a great deal of attention as a primary group of candidates for many biomedical applications such as resorbable and non-resorbable ceramics, cements, drug carriers, prosthetic coatings, and composite materials for bone reconstruction and replacement, bone defect-filling, drug carrier, and coatings of metal prostheses 1,2 . The incorporation of various atoms in both cation and anion sites has been studied to improve the structural, physical, and chemical properties of CaPs to sustain thermal stability, control dissolution, obtain antibacterial property, and improve osteoblast differentiation and osteoclast proliferation 3-7 . Among these CaPs, particular attention has been given to β-tricalcium phosphate (Ca 3 (PO 4 ) 2 , β-TCP) owing to its resorbability in vivo accompanied by new bone growth and replacement 8,9 .
-TCP has a rhombohedral structure with a space group of R3c, described by A and B columns running along the c-axis (Supp. Fig. S1) 10 . The structure of -TCP allows ionic replacements, especially the incorporation of cations at the calcium sites in the A columns [11][12][13] . Calcium ions in β-TCP are present in five locations. Ca(1), Ca (2), and Ca (3)  Ag incorporation in CaPs has been investigated to achieve antibacterial activity by the release of Ag atoms in the human body [16][17][18][19][20][21][22][23][24] . However, to date, due to the limitations of analytical techniques, very few experimental studies have provided quantitative information on capacity of Ag incorporation into β-TCP structure 25 . It is essential to elucidate the limit of incorporation of Ag atoms in the β-TCP structure with respect to the incorporation sites.
To understand the mechanism of the incorporation of atoms in β-TCP and characterize the naturally-and/or pathologically-incorporated elements in CaPs, it is necessary to perform detailed structural characterization of such CaPs.
Previously, ion incorporations in CaP structures have been investigated with the help of Xray diffraction (XRD) and neutron diffraction refined by the Rietveld method, Raman spectroscopy, ab-initio calculations, nuclear magnetic resonance, and first-principle calculations [26][27][28][29][30][31][32][33] . Yoshida et al. estimated the monovalent incorporation in either Ca (4) or Ca(5) sites of β-TCP with the help of lattice parameter calculations 14 . Matsumoto et al. claimed that the amount of Ag incorporation is limited to 9.09 at% with possible incorporation in Ca(4) sites and vacancies, whereas, smaller Zn ions may be incorporated in half of the Ca(5) sites simultaneously 20 . However, comprehensive studies have not been performed to reveal the structure of an Ag-incorporated β-TCP structure.
High-resolution transmission electron microscopy (HRTEM) is a powerful tool to directly observe individual incorporation at the atomic scale. However, conventional HRTEM can easily damage the β-TCP structure because of the beam-sensitive nature of the structure 34 .
In this study, the estimated locations of Ag incorporation were assessed by Rietveld refinements and proven using aberration-corrected high-angle annular dark-field (HAADF)-STEM (Cs-corrected HAADF-STEM). Cs-corrected STEM has an advantage over the traditional TEM because of its lesser image degradation 35 . Two common approaches for reducing the damage caused by electron dosage in STEM are decreasing the pixel dwell time and lowering the beam current density, which are parameters that can be adjusted 36 . Cs-corrected STEM is capable of rapid scanning while avoiding possible damage during observation and simultaneously generating sub-angstrom resolution 37 . It has been reported that the HAADF-STEM signal is proportional to the square of the atomic number of the element, which allows an atom-by-atom chemical identification 38 , such as of the Ag atom in the β-TCP structure, the atomic number of Ag being higher than those of Ca and P atoms. This study focuses on using low-dose STEM image recording with the wideangle data collection of the annular dark-field detector, which is advantageous for achieving a high signal/noise ratio of measurement to reach high resolution capability of the instrument 39 . With the help of the state-of-art HAADF-STEM observations and corresponding image simulations, we have provided an atomic insight into the location of Ag in the β-TCP structure, which is stabilized by incorporation of Ag atoms at specific Ca sites. To the authors' best knowledge, the present study is the first evidence of ionic incorporation in β-TCP performed with the state-of-art HAADF-STEM.
In the previous studies, we had clarified that the incorporation of Ag atoms stabilized the structure with a decrease in the lattice parameters, resulting in a suppression of β-to-α transformation of TCP, an improvement in thermal stability, and a decrease in bioresorbability while also exhibiting effective antibacterial activity 3, 40 . The alteration in the properties of β-TCP highlights the importance of the limit of Ag incorporation at the various sites of the β-TCP structure. Further, Ag incorporation can also affect the bonding of CaP coatings on metal implant materials because of increased charge transfer between the coating and metal implant 41 . Therefore, the properties of CaP coatings altered with ion incorporation [42][43][44] may be correlated to the findings of this study. The present study, for the first time, investigates the incorporation of Ag in Ca(4) and Ca(5) site positions of the β-TCP structure and estimates a possible Ag incorporation limit with the help of an atom-byatom structural evaluation by using the state-of-art HAADF-STEM observations and corresponding simulations.

Results
The Ag-incorporated -TCP with an Ag/(Ca+Ag) atomic ratio of 0.091 (0.09Ag-TCP), which is considered to be the theoretical limit of monovalent incorporation for Ca(4) sites 20 , was used to locate the Ag atoms in the Ca(4) sites of -TCP. The Ag-incorporated -TCP with an Ag/(Ca+Ag) atomic ratio of 0.291 (0.29Ag-TCP), which exceeds the theoretical limit, was investigated to access the experimental limit of Ag incorporation in -TCP structure.

Distribution of Ag atoms in β-TCP structure
EELS scanning (50 × 50 nm) was used to observe the distribution of Ag atoms in the β-TCP structure, as shown in Fig. 1. The signals related to Ca, P, and O were detected homogeneously, indicating the presence of regular atoms in the β-TCP structure. Further, the Ag signal was well resolved and uniformly dispersed in the β-TCP matrix with no clustering and agreed with the TEM-EDS line analysis (Supp. Fig. S2). Even though the Ag incorporation was proven, information about the positions of the Ag atoms in the β-TCP structure was lacking. To confirm the position of the localized Ag atoms in the β-TCP structure, high-resolution TEM-EDS was conducted at a resolution of 10 × 10 nm (Supp.

Structural model of Ag incorporation in β-TCP
The crystal structure model of the Ag-incorporated β-TCP is presented in Fig. 2(a); the Ca(4) sites and vacancies are occupied by Ag atoms, shown as red spheres with half occupancy. The crystal structure of β-TCP can be described by A and B columns along the c-axis (Supp. Fig. S1). The crystal structure of Ag-incorporated β-TCP along the [001] and [010] directions is presented in Fig. 2(b) and 2(c), respectively, to display the two columns.
Column A, containing Ca(4) site and Ca(5) site atoms, has a unique atomic arrangement.

Rietveld refinement of Ag incorporation in β-TCP
To further investigate the Ag incorporation in β-TCP structure, an XRD pattern of pure β-TCP after sintering at 1273 K was obtained, and Rietveld analysis was performed on this pattern ( Fig. 3(a)). The structural parameters obtained were in good agreement with the calculated patterns. The β-TCP structural parameters reported in the literature 10 were used as the initial parameters in the Rietveld analysis. The refined structural and profile parameters of pure β-TCP were employed for the Rietveld analysis of the Ag-incorporated β-TCP structure. The effect of Ag atom incorporation on the β-TCP structure was investigated for 0.09Agβ-TCP and 0.29Agβ-TCP.
When a higher ratio of Ag (charged atomic ratio of Ag/(Ca+Ag) = 0.29) was reacted with β-TCP, metallic Ag and Ag 3 PO 4 secondary phases were detected, indicating that the incorporation limit of Ag atoms in the β-TCP structure was reached (Fig. 3(c)). The measured atomic ratio for 0.29Agβ-TCP was Ag/(Ca+Ag) = 0.12 (Table 1), which was slightly higher than the incorporation limit to Ca(4) sites and Ca (4)

HAADF-STEM of the [001] zone axis of Ag incorporation in β-TCP
The atomic-resolution HAADF-STEM was carried out for the 0.29Agβ-TCP and pure β-TCP at the [001] incident beam axis corresponding to zone axis reflections of electron diffraction (ED) patterns (Fig. S4). The ED pattern corresponds to rhombohedral β-TCP  46 . with the atomic structure model (Fig. 4(c)), suggesting that bright Ag atoms on the A column of the β-TCP structure were positioned in the hexagonal structure. Meanwhile, Ca atoms on B column exhibited a blurry region between the Ag atoms located on the A column, corresponding to the Rietveld refinement as shown in Fig 3(b, c). Although  Fig. 4(a), exhibiting higher and lower z-values as shown in Fig. 4(e), and indicating the atoms with a higher atomic number (Ag) in A column and lower atomic number (Ca) in B column. In contrast, the P atoms were equally located in both columns.
To our best knowledge, this is the first time in literature that the state-of-the-art aberrationcorrected HAADF-STEM observations and corresponding multislice simulations were carried out to detect the location of incorporated atoms in CaPs, which is challenging owing to its complex and radiation-sensitive structure.
IFFT HAADF-STEM of pure β-TCP at the [001] incident beam axis was evaluated for comparison with the Ag-incorporated β-TCP structure, as shown in Fig. 4

HAADF-STEM of the [010] zone axis of Ag incorporation in β-TCP
HAADF-STEM observation for the [010] zone axis of 0.29Agβ-TCP and pure β-TCP structure (Fig. S5), which is along the c-axis, was carried out to confirm the atomic position of the excess amount of Ag atoms, which was more than the theoretical limit of the Ca (4) sites. Fig. 5(a) shows the IFFT HAADF-STEM image of the [010] zone axis confirmed with ED ( Fig. 5(b)) that significantly resolved the arrangement of Ag atoms in the A columns of the β-TCP structure, and is illustrated in the structural model (Fig. 5(c)). The   Fig. 5(f, h, i). The z-contrast profile (Fig. 5(j)) exhibits Ca atoms with higher intensity due to the high-density Ca atomic locations, shown within rectangles in Fig. 5(f, h). No significant studies focusing on the structure of Ag-incorporated β-TCP have been reported. Among the few investigations that have been reported, the most relevant study is by Matsumoto et al. 20 , who reported that the maximum incorporation of monovalent metal ions for β-TCP was an Ag/(Ca+Ag) atomic ratio of 0.909 for Ca(4) site, including vacancies 20 . However, in the present study, the Ag incorporation was measured to have reached its limit at an Ag/(Ca+Ag) atomic ratio of 0.12 in the β-TCP matrix of 0.29Ag-TCP in this study (Table 1) (1) 2 + = 2 + + (4) Where V Ca(4) is the vacancy of Ca(4) site. Therefore, all Ca(4) sites including vacancies in the β-TCP structure could be occupied by the Ag + ions, resulting in a decrease in the lattice parameter c (Fig. 3(d)) and stabilization of the β-TCP structure. The structural stabilization due to the Ag incorporation has been reported to improve thermal stability and delay dissolution of the β-TCP 3,51 . However, there is no structural confirmation of this incorporation or microscopic evidence regarding its location in the β-TCP structure. Hence, it is scientifically important to confirm the location of incorporation in the β-TCP structure for relating the structural limit of incorporation and its effect on the properties of the β-TCP structure 52 . This confirmation is the novelty of this study.

Discussion
To determine the location of excess Ag after incorporation in the Ca(4) sites, approximately 15 % of the Ca(5) sites were investigated at the more complex [010] zone axis (Fig. 5). The results suggested that Ag atoms were not systematically incorporated in the Ca(5) sites. In this study, Ag atoms exceeding the theoretical limit of Ca(4) site incorporation were assumed to be irregularly located at Ca (5)

Conclusion
In this study, the crystallographic investigation of Ag incorporation in β-TCP structure with

XRD analysis
The phases and lattice parameters of sintered compacts were determined using XRD (Ultima IV, Rigaku Corp., Tokyo, Japan) with Cu Kα radiation (λ = 1.54184 Å). Phase analysis and Rietveld refinement were performed with X'Pert HighScore and FullProf, respectively, on XRD patterns of the Ag-incorporated β-TCP structure, comparing with the standard β-TCP structure (JCPDS#9-169). Data used for structural investigation were collected in the 2θ range 10°-70° with a 0.02° step and a counting time of 200 s/step.

Atomic-scale evaluation using HAADF-STEM
Gatan PIPS ion milling and plasma cleaning (SOLARUS Gatan) were used for the thinning and cleaning of sintered compacts, respectively. Ion milling was conducted with a low energy electron beam at an angle of 4° to produce an electron-transparent TEM specimen.