Bimodal Ultrasound and X-ray Bioimaging Properties of Particulate Calcium Fluoride Biomaterial

Ultrasound (US) and X-ray imaging are diagnostic methods that are commonly used to image internal body structures. Several organic and inorganic imaging contrast agents are commercially available. However, their synthesis and purification remain challenging, in addition to posing safety issues. Here, we report on the promise of widespread, safe, and easy-to-produce particulate calcium fluoride (part-CaF2) as a bimodal US and X-ray contrast agent. Pure and highly crystalline part-CaF2 is obtained using a cheap commercial product. Scanning electron microscopy (SEM) depicts the morphology of these particles, while energy-dispersive X-ray spectroscopy (EDS) confirms their chemical composition. Diffuse reflectance ultraviolet-visible spectroscopy highlights their insulating behavior. The X-ray diffraction (XRD) pattern reveals that part-CaF2 crystallizes in the face-centered cubic cell lattice. Further analyses regarding peak broadening are performed using the Scherrer and Williamson–Hall (W-H) methods, which pinpoint the small crystallite size and the presence of lattice strain. X-ray photoelectron spectroscopy (XPS) solely exhibits specific peaks related to CaF2, confirming the absence of any contamination. Additionally, in vitro cytotoxicity and in vivo maximum tolerated dose (MTD) tests prove the biocompatibility of part-CaF2. Finally, the results of the US and X-ray imaging tests strongly signal that part-CaF2 could be exploited in bimodal bioimaging applications. These findings may shed a new light on calcium fluoride and the opportunities it offers in biomedical engineering.


Introduction
Currently, ultrasound (US) echography (also known as sonography) and X-ray radiography are among the most frequently employed techniques in bioimaging [1][2][3]. Several materials have been designed to serve as contrast agents to improve the quality of the acquired images. In the case of X-ray imaging, iodine-and barium sulfate-based compounds 2. Results and Discussion 2.1. Physicochemical Characterization 2. 1

.1. Scanning Electron Microscopy Analysis
We describe a simple and cheap method to obtain high-purity, crystalline particulate calcium fluoride (part-CaF 2 ) starting from a commercially available sealant for applications as a bimodal contrast agent in ultrasound and X-ray bioimaging. SEM analysis of the resulting white powder showed particles of irregular shapes and different sizes, where the size distribution ranged from a few hundred nanometers to a few microns ( Figure 1A,B). However, the size of most of the particles spanned from 500 nm to 2 µm. Although the optimum size depends on the targeted bioapplications [9], the part-CaF 2 generated in this study fell within the range of liposomes and polymeric particles used in drug delivery systems and biomedical imaging [9,10]. Moreover, various methods enabled the size control of the CaF 2 particles to fit a specific application [16,20,24,28]. Additionally, EDX analysis showed the existence of various chemical elements at different concentrations, including calcium and fluorine ( Figure 1C). However, the presence of carbon and oxygen may be ignored, as they most likely come from the adhesive layer used to prepare the sample for analysis. By dividing the molar percentages, a fluorine-to-calcium ratio of 2:1 was obtained, which fits the CaF 2 chemical formula.

X-ray Diffraction
The XRD pattern of part-CaF2 (Figure 2A) displays typical peaks of pure CaF2 crystallizing in the face-centered cubic cell lattice (JCPDS Card no 87-0971) corresponding to the Fm3m crystalline group, in excellent agreement with published data ( Figure 2B) [19,29]. Interestingly, the absence of extra peaks depicts the high purity of the obtained sample (cf. XPS). Prior to determining the cell lattice parameter, a Lorentzian function was applied for fitting purposes as it fits the XRD profile better when compared to the Gaussian analog (Insert Figure 2A). Using Equation (2), the lattice parameter, a, was determined to be 5.4615 Å, corroborating previously reported data [29][30][31]. Additionally, the index of crystallinity (IC) of part-CaF2 was estimated using Equation (3) to be 93.26%, denoting its high degree of crystallinity.

X-ray Diffraction
The XRD pattern of part-CaF 2 (Figure 2A) displays typical peaks of pure CaF 2 crystallizing in the face-centered cubic cell lattice (JCPDS Card no 87-0971) corresponding to the Fm3m crystalline group, in excellent agreement with published data ( Figure 2B) [19,29]. Interestingly, the absence of extra peaks depicts the high purity of the obtained sample (cf. XPS). Prior to determining the cell lattice parameter, a Lorentzian function was applied for fitting purposes as it fits the XRD profile better when compared to the Gaussian analog (Insert Figure 2A). Using Equation (2), the lattice parameter, a, was determined to be 5.4615 Å, corroborating previously reported data [29][30][31]. Additionally, the index of crystallinity (IC) of part-CaF 2 was estimated using Equation (3) to be 93.26%, denoting its high degree of crystallinity.
for analysis. By dividing the molar percentages, a fluorine-to-calcium ratio of 2:1 was obtained, which fits the CaF2 chemical formula.

X-ray Diffraction
The XRD pattern of part-CaF2 ( Figure 2A) displays typical peaks of pure CaF2 crystallizing in the face-centered cubic cell lattice (JCPDS Card no 87-0971) corresponding to the Fm3m crystalline group, in excellent agreement with published data ( Figure 2B) [19,29]. Interestingly, the absence of extra peaks depicts the high purity of the obtained sample (cf. XPS). Prior to determining the cell lattice parameter, a Lorentzian function was applied for fitting purposes as it fits the XRD profile better when compared to the Gaussian analog (Insert Figure 2A). Using Equation (2), the lattice parameter, a, was determined to be 5.4615 Å, corroborating previously reported data [29][30][31]. Additionally, the index of crystallinity (IC) of part-CaF2 was estimated using Equation (3) to be 93.26%, denoting its high degree of crystallinity.   The determination of the crystallite size of part-CaF 2 ( Figure 2B) was carried out using low-angle and high-angle peaks and Equation (5). Nonetheless, low-range analysis provided more precise results because higher angles cause distortion and hence poor precision [32]. part-CaF 2 exhibited a relatively small crystallite size of about 8-14 nm, values that were slightly smaller than those reported in published data [29,33]. Besides, the average crystallite size was significantly smaller than the particle size provided by SEM micrographs (Cf. Figure 1), demonstrating that the part-CaF 2 sample was polycrystalline. Using ultrasonic probes or ball milling, it was possible to tune the particle size and downsize it to values approaching the crystallite size by breaking down the agglomerates bonded by van der Waals (vdW), capillary, or electrostatic interactions to give rise to a homogenous sample of much smaller particles that might be considered as monocrystalline [34][35][36].

Diffuse Reflectance UV-Vis Spectroscopy
The reflectance of part-CaF 2 was recorded to study its optical properties ( Figure 3). It clearly shows that the reflectance of part-CaF 2 was ca. 55% in the lower limit of the spectrum at λ = 260 nm and increased steadily in the visible region without plateauing nor reaching its maximum value, as the recorded value at 800 nm was smaller than 75%. In other words, part-CaF 2 partially absorbed the incident radiations in either the UV or visible regions. Moreover, the reflectance spectrum displayed weak peaks pointing down at 377.7 nm, 448.3 nm, and 622.7 nm, which may arise from the large surface-to-volume ratio exhibited by part-CaF 2 [29]. Besides, the intense peak, observed at 260 nm, was attributed to surface defect absorption, such as Schottly and Frenkel, dangling bonds, and regions of disorder, which are common in particles [29,37]. On the other hand, the Kubelka-Munk (K-M) transformation of part-CaF 2 did not permit the determination of the band gap since it is found in the high-ultraviolet region at around 10 eV [38]. However, part-CaF 2 may be considered as a promising insulator, with a lattice constant similar to silicon due to its very large band gap [39].
The determination of the crystallite size of part-CaF2 ( Figure 2B) was carried out using low-angle and high-angle peaks and Equation (5). Nonetheless, low-range analysis provided more precise results because higher angles cause distortion and hence poor precision [32]. part-CaF2 exhibited a relatively small crystallite size of about 8-14 nm, values that were slightly smaller than those reported in published data [29,33]. Besides, the average crystallite size was significantly smaller than the particle size provided by SEM micrographs (Cf. Figure 1), demonstrating that the part-CaF2 sample was polycrystalline. Using ultrasonic probes or ball milling, it was possible to tune the particle size and downsize it to values approaching the crystallite size by breaking down the agglomerates bonded by van der Waals (vdW), capillary, or electrostatic interactions to give rise to a homogenous sample of much smaller particles that might be considered as monocrystalline [34][35][36].

Diffuse Reflectance UV-Vis Spectroscopy
The reflectance of part-CaF2 was recorded to study its optical properties ( Figure 3). It clearly shows that the reflectance of part-CaF2 was ca. 55% in the lower limit of the spectrum at λ = 260 nm and increased steadily in the visible region without plateauing nor reaching its maximum value, as the recorded value at 800 nm was smaller than 75%. In other words, part-CaF2 partially absorbed the incident radiations in either the UV or visible regions. Moreover, the reflectance spectrum displayed weak peaks pointing down at 377.7 nm, 448.3 nm, and 622.7 nm, which may arise from the large surface-to-volume ratio exhibited by part-CaF2 [29]. Besides, the intense peak, observed at 260 nm, was attributed to surface defect absorption, such as Schottly and Frenkel, dangling bonds, and regions of disorder, which are common in particles [29,37]. On the other hand, the Kubelka-Munk (K-M) transformation of part-CaF2 did not permit the determination of the band gap since it is found in the high-ultraviolet region at around 10 eV [38]. However, part-CaF2 may be considered as a promising insulator, with a lattice constant similar to silicon due to its very large band gap [39].  Figure 4A displays the XPS general survey spectrum of part-CaF2 calibrated at C1s in C-C/C-H with a binding energy (BE) of 285.0 eV. It clearly exhibits two prominent peaks arising from the two elements constituting this sample, that is, fluorine (F1s) and calcium (Ca2p); it also exhibits less intense peaks related to carbon (C1s) and oxygen (O1s). The C1s peak can be deconvoluted into its 3 components, C-C/C-H, C-O, and C=O, centered at 285.0 eV, 285.7 eV, and 288.8 eV, respectively ( Figure 4E) [40]. The C1s peak most likely  Figure 4A displays the XPS general survey spectrum of part-CaF 2 calibrated at C1s in C-C/C-H with a binding energy (BE) of 285.0 eV. It clearly exhibits two prominent peaks arising from the two elements constituting this sample, that is, fluorine (F1s) and calcium (Ca2p); it also exhibits less intense peaks related to carbon (C1s) and oxygen (O1s). The C1s peak can be deconvoluted into its 3 components, C-C/C-H, C-O, and C=O, centered at 285.0 eV, 285.7 eV, and 288.8 eV, respectively ( Figure 4E) [40]. The C1s peak most likely originated from a background source, i.e., the tape used to mount the sample onto the XPS holder [30,36]. On the other hand, the deconvolution of the peak O1s revealed the presence of signals that correspond to O 2− (532.7 eV) and OH − (535.4 eV) due to the well-known adsorption of H 2 O molecules on the CaF 2 (111) plane ( Figure 4C) [30,36]. This fact is also reflected in the high-resolution F1s peak ( Figure 4B) that displays a very prominent peak centered at 684.9 eV, assigned to F-Ca bonding, in addition to a very weak peak centered at 686.9 eV that is due to F − defects at the surface of CaF 2 , most likely originating from water adsorption [28,30,36]. Moreover, the Ca2p peak displayed a doublet owing to the spin-orbit splitting typical of Ca(II) assigned to Ca2p 1/2 and Ca2p 3/2 , centered at 351.4 eV and 348.0 eV, respectively, and with 1:2 area ratio ( Figure 4D) [28,30,36,[40][41][42][43]. Finally, the XPS analysis enabled the estimation of the F:Ca atomic ratio to equal 2.04, highlighting the chemical formula of CaF 2 and corroborating the SEM-EDX findings.

X-ray Photoelectron Spectroscopy
Molecules 2021, 26, x FOR PEER REVIEW 5 of 13 originated from a background source, i.e., the tape used to mount the sample onto the XPS holder [30,36]. On the other hand, the deconvolution of the peak O1s revealed the presence of signals that correspond to O 2− (532.7 eV) and OH -(535.4 eV) due to the well-known adsorption of H2O molecules on the CaF2 (111) plane ( Figure 4C) [30,36]. This fact is also reflected in the high-resolution F1s peak ( Figure 4B) that displays a very prominent peak centered at 684.9 eV, assigned to F-Ca bonding, in addition to a very weak peak centered at 686.9 eV that is due to Fdefects at the surface of CaF2, most likely originating from water adsorption [28,30,36]. Moreover, the Ca2p peak displayed a doublet owing to the spin-orbit splitting typical of Ca(II) assigned to Ca2p1/2 and Ca2p3/2, centered at 351.4 eV and 348.0 eV, respectively, and with 1:2 area ratio ( Figure 4D) [28,30,36,[40][41][42][43]. Finally, the XPS analysis enabled the estimation of the F:Ca atomic ratio to equal 2.04, highlighting the chemical formula of CaF2 and corroborating the SEM-EDX findings.

In Vitro Cytotoxicity
The in vitro toxicity of various concentrations of part-CaF2 against 3T3 fibroblasts was studied via MTT assay ( Figure 5). Compared to the control group, the 3T3 fibroblasts maintained their full viability (90-100%) for part-CaF2 concentrations of up to 2000 μg·mL −1 and 60% at the highest tested concentration (3000 μg·mL −1 ). These results highlight the excellent biocompatibility exhibited by these particles, making them suitable candidates for numerous applications in the biomedical field. The in vitro toxicity of various concentrations of part-CaF 2 against 3T3 fibroblasts was studied via MTT assay ( Figure 5). Compared to the control group, the 3T3 fibroblasts maintained their full viability (90-100%) for part-CaF 2 concentrations of up to 2000 µg·mL −1 and 60% at the highest tested concentration (3000 µg·mL −1 ). These results highlight the excellent biocompatibility exhibited by these particles, making them suitable candidates for numerous applications in the biomedical field. originated from a background source, i.e., the tape used to mount the sample onto the XPS holder [30,36]. On the other hand, the deconvolution of the peak O1s revealed the presence of signals that correspond to O 2− (532.7 eV) and OH -(535.4 eV) due to the well-known adsorption of H2O molecules on the CaF2 (111) plane ( Figure 4C) [30,36]. This fact is also reflected in the high-resolution F1s peak ( Figure 4B) that displays a very prominent peak centered at 684.9 eV, assigned to F-Ca bonding, in addition to a very weak peak centered at 686.9 eV that is due to Fdefects at the surface of CaF2, most likely originating from water adsorption [28,30,36]. Moreover, the Ca2p peak displayed a doublet owing to the spin-orbit splitting typical of Ca(II) assigned to Ca2p1/2 and Ca2p3/2, centered at 351.4 eV and 348.0 eV, respectively, and with 1:2 area ratio ( Figure 4D) [28,30,36,[40][41][42][43]. Finally, the XPS analysis enabled the estimation of the F:Ca atomic ratio to equal 2.04, highlighting the chemical formula of CaF2 and corroborating the SEM-EDX findings.

In Vitro Cytotoxicity
The in vitro toxicity of various concentrations of part-CaF2 against 3T3 fibroblasts was studied via MTT assay ( Figure 5). Compared to the control group, the 3T3 fibroblasts maintained their full viability (90-100%) for part-CaF2 concentrations of up to 2000 μg·mL −1 and 60% at the highest tested concentration (3000 μg·mL −1 ). These results highlight the excellent biocompatibility exhibited by these particles, making them suitable candidates for numerous applications in the biomedical field.

Determination of Maximum Tolerated Dose
In the first methodology, the initial dose for each animal started at 1.00 mg·kg −1 and all the doses were staggered up to 33.79 mg·kg −1 ( Figure 6A1). In the second methodology (based on the ICH M3-R2), the initial dose for each animal started at 1 mg·kg −1 , up to the maximum dose of 10.00 mg·kg −1 ( Figure 6A2). The test was stopped on day 10 for both methodologies since the animals had not shown any notable variation in the parameters described in Table 1 (such as weight loss over 20%), or because none of the scores were over 2, as clearly depicted in Figure 6A2,B2. For both methodologies, the weight of all animals increased, and no clinical signs were observed ( Figure 6). Therefore, it is possible to affirm that part-CaF 2 is safe up to the dose of 33.79 mg·kg −1 . Due to limitations in the amount of material produced, it was not possible to check the maximum tolerated dose (MTD) itself. However, even a very high dose shows no adverse effect on the animals.

Determination of Maximum Tolerated Dose
In the first methodology, the initial dose for each animal started at 1.00 mg·kg −1 and all the doses were staggered up to 33.79 mg·kg −1 ( Figure 6A1). In the second methodology (based on the ICH M3-R2), the initial dose for each animal started at 1 mg·kg −1 , up to the maximum dose of 10.00 mg·kg −1 ( Figure 6A2). The test was stopped on day 10 for both methodologies since the animals had not shown any notable variation in the parameters described in Table 1 (such as weight loss over 20%), or because none of the scores were over 2, as clearly depicted in Figure 6(A2,B2). For both methodologies, the weight of all animals increased, and no clinical signs were observed ( Figure 6). Therefore, it is possible to affirm that part-CaF2 is safe up to the dose of 33.79 mg·kg −1 . Due to limitations in the amount of material produced, it was not possible to check the maximum tolerated dose (MTD) itself. However, even a very high dose shows no adverse effect on the animals.

Ultrasound and X-ray Imaging
The ultrasound (US) imaging test, performed by subjecting an Eppendorf tube filled with part-CaF 2 ( Figure 7A) to US irradiation, demonstrated the US absorption properties of part-CaF 2 . In this image, part-CaF 2 was suspended in water to reproduce a tissue environment (performed by casting the part-CaF 2 into a flat petri dish), showing that US Molecules 2021, 26, 5447 7 of 13 attenuation was exclusively generated by part-CaF 2 reaching almost its maximum intensity. US attenuation occurs with sonosensitizers when the cavitation, followed by the collapse of small bubbles, produces enough energy to induce heating [6]. Although the mechanism of sonosensitizer excitation is not fully understood, most molecules exhibiting US attenuation contain halogen atoms in their structure [6], a common feature shared by commercially available US contrast agents [8], polytetrafluorethylene [11], and the part-CaF 2 described in the present work. Thus, the results suggest that fluorine atoms from part-CaF 2 likely contribute to the US absorption. However, the presence of halogen atoms in the molecular structure is not a requisite for US attenuation by a given material, as reported recently [44]. To the best of our knowledge, this is the first report showing the US imaging properties of part-CaF 2 materials. These outstanding results may elicit interest in calcium fluoride for use in biomedical imaging applications.
The ultrasound (US) imaging test, performed by subjecting an Eppendorf tube filled with part-CaF2 ( Figure 7A) to US irradiation, demonstrated the US absorption properties of part-CaF2. In this image, part-CaF2 was suspended in water to reproduce a tissue environment (performed by casting the part-CaF2 into a flat petri dish), showing that US attenuation was exclusively generated by part-CaF2 reaching almost its maximum intensity. US attenuation occurs with sonosensitizers when the cavitation, followed by the collapse of small bubbles, produces enough energy to induce heating [6]. Although the mechanism of sonosensitizer excitation is not fully understood, most molecules exhibiting US attenuation contain halogen atoms in their structure [6], a common feature shared by commercially available US contrast agents [8], polytetrafluorethylene [11], and the part-CaF2 described in the present work. Thus, the results suggest that fluorine atoms from part-CaF2 likely contribute to the US absorption. However, the presence of halogen atoms in the molecular structure is not a requisite for US attenuation by a given material, as reported recently [44]. To the best of our knowledge, this is the first report showing the US imaging properties of part-CaF2 materials. These outstanding results may elicit interest in calcium fluoride for use in biomedical imaging applications. Additionally, part-CaF2 exhibits X-ray attenuation, which is supported by the contrast signal observed in Figure 7B. Indeed, part-CaF2 neatly attenuates X-rays of different energies (i.e., different potentials applied at the same intensity). Moreover, the same material was subjected to different parameters by tuning the tube current and potential without affecting X-ray attenuation. This result highlights the capability of part-CaF2 to absorb the incident electromagnetic radiation of high energy X-rays at different conditions without affecting the yield.
As a contrast agent for both US and X-ray imaging, CaF2 may offer tremendous advantages when compared to analog molecules and particles either already used or in development for the same purpose [5,7,8]. In fact, this material is widespread in nature as a mineral. It is cheap and easy to produce via various methodologies [29,[45][46][47], and may be water-soluble [48]. This should make its large-scale production easily achievable at very competitive costs. Besides, it has been already demonstrated that this biomaterial is amenable to entrapment within hydrogels [17,20], polymers [26], and resins [46,49]. All these facts presage an easy and versatile surface chemistry aiming at greater targeting efficiency and even increased biocompatibility, thus paving the way for other clinical uses in the biomedical field. Additionally, part-CaF 2 exhibits X-ray attenuation, which is supported by the contrast signal observed in Figure 7B. Indeed, part-CaF 2 neatly attenuates X-rays of different energies (i.e., different potentials applied at the same intensity). Moreover, the same material was subjected to different parameters by tuning the tube current and potential without affecting X-ray attenuation. This result highlights the capability of part-CaF 2 to absorb the incident electromagnetic radiation of high energy X-rays at different conditions without affecting the yield.
As a contrast agent for both US and X-ray imaging, CaF 2 may offer tremendous advantages when compared to analog molecules and particles either already used or in development for the same purpose [5,7,8]. In fact, this material is widespread in nature as a mineral. It is cheap and easy to produce via various methodologies [29,[45][46][47], and may be water-soluble [48]. This should make its large-scale production easily achievable at very competitive costs. Besides, it has been already demonstrated that this biomaterial is amenable to entrapment within hydrogels [17,20], polymers [26], and resins [46,49]. All these facts presage an easy and versatile surface chemistry aiming at greater targeting efficiency and even increased biocompatibility, thus paving the way for other clinical uses in the biomedical field.

Preparation of Particulate CaF 2
In this step, 0.3 g of a gray pipe thread compound (ACE ® ) and 2 mL of 40% aq. hydrofluoric acid (HF) were mixed in a Falcon tube and subjected to sonication for 2 h in an ultrasonic bath. As a result, 3 phases appeared. In addition to a dark gray substance (excipients) at the top and a translucent supernatant as the intermediate phase, the bottom phase consisted of a precipitate. This pellet, made of particulate CaF 2 (part-CaF 2 ), was collected and mixed with another 2 mL of 40% aq. HF in a Falcon tube and subjected to sonication for 45 min in an ultrasonic bath. The sample was then centrifuged at 2000 rpm for 5 min. Finally, the pellet of part-CaF 2 was collected and washed 3 times using isopropyl alcohol, dried in an oven at 80 • C for 3 h, and stored for further analysis and investigation.
3.2. Physico-chemical Characterization 3.2.1. Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy (SEM-EDX) The scanning electron microscopy analysis of the part-CaF 2 sample was carried out using a TESCAN FEG SEM MIRA3 apparatus. part-CaF 2 powder was fixed onto SEM stubs for analysis using a carbon adhesive layer and sputter-coated with an approximately 20 nm gold (99.99% purity) layer. The EDX analysis was performed using the same device since it is equipped with a Bruker X-Flash 6|30 detector, with 123 eV resolution at Mn Kα with an EDX detector.

X-ray Diffraction
X-ray diffraction analysis was carried out on the part-CaF 2 sample using a PANalytical brand θ-2θ configuration (Bragg-Brentano geometry) X-ray tube, with Cu Kα irradiation λ = 1.54059 Å in an EMPYREAN diffractometer. The acquired XRD pattern was fitted using OriginPro software to determine the lattice parameters, index of crystallinity, and crystallite size. The interplanar distance, d, was determined using Bragg's law (Equation (1)).
where n is the order of reflection (n = 1), and λ is the wavelength of Cu Kα irradiation. Then, the main peaks were compared with reliable databases to assign Miller indices, (hkl), where a face-centered cubic crystal system for CaF 2 was determined for which the space group was Fm3m. Therefore, there were 3 equal axes at right angles, for which the lattice parameter value was computed using Equation (2): The lattice parameter a was calculated using the interplanar distance of the most intense peak (111). Besides, the index of crystallinity (IC) of the part-CaF 2 sample was determined by comparing the ratios between the areas of crystalline peaks (crystalline phase) and of their respective background (amorphous phase) using the empirical Equation (3): In addition, the crystallite size was determined by analyzing the breadth peak. Instrumental and physical broadening effects were considered as broadening of the Bragg peaks (Equation (4)) [50]: The broadening due to the instrumental setup was corrected with a previous diffraction pattern using a silicon standard specimen. Then, substitution of Equation (4) into the well-known Scherrer equation yields the following formulation (Equation (5)): where β hkl is the breadth value of the full width at half maximum (FWHM) taken on a 2θ scale (transformed into radians); K is a numerical constant equal to 0.94; θ is the Bragg angle taken on a 2θ scale (in degrees); λ = 1.54059 Å; and L is the linear dimension of the particle or crystallite size. The crystallite size is the average of values calculated for each diffraction peak.
Further details regarding the XRD analysis of part-CaF 2 powder are provided in the Supporting Information (SI).

Diffuse Reflectance UV-Vis Spectroscopy
Diffuse Reflectance UV-Vis spectroscopy was performed on part-CaF 2 powder to determine its optical characteristics. The "LAMBDA 1050 UV-Vis Spectrophotometer PerkinElmer ® , equipped with a PerkinElmer ® accessory 3D WB Detector Module and the Praying Mantis™ Diffuse Reflection Accessory, was employed. The light spot was about 1-2 mm. The powder was placed in the sample holder (a hole of 10 mm in diameter and 3 mm depth) and the surface was flattened. A white standard of BaSO 4 was used as blank prior to measurement. Band gap analysis was carried out using the Kubelka-Munk (K-M) function [11]. The x-axis (wavelength) was converted to energy, E, by applying the Einstein-Planck relation (Equation (6)): where h is the Planck constant (4.135667 × 10 −15 eV), c is the speed of light, and λ is the wavelength. The y-axis (reflectance) was converted to [k/s·hν] 2 by applying the Kubelka-Munk function (Equation (7)): where k is the absorption coefficient, s is the scattering coefficient, and R is the reflectance. The value of the band gap was determined graphically by extrapolating a straight line at k = 0.

X-ray Photoelectron Spectroscopy (XPS)
The XPS spectrum of the part-CaF 2 sample was recorded using a Thermo VG ES-CALAB 250 (East Grinstead, UK) fitted with a monochromated Al Kα X-ray source with an incident energy of 1486.6 eV. An electron flood gun was used for charge compensation. The analyzer was operated at 40 and 100 eV pass energy for the narrow regions and survey spectra, respectively. Elemental atomic concentrations were calculated from the XPS peak areas and the corresponding Scofield sensitivity factors corrected for the analyzer transmission work function. The measurement was conducted at 255 eV survey operation pass energy and 55 eV for high-resolution narrow regions. Prior to the analysis, the recorded spectrum was calibrated with C1s peak in C-C/C-H at 285.0 eV, followed by fitting and deconvolution of the relevant XPS peaks using the Gauss-Lorentzian function [40,43].

Biological Properties of Part-CaF 2 3.3.1. In Vitro Cytotoxicity
This was carried out using a routine assay that is commonly used to study the toxicity of nanomaterials. Namely, 3T3 fibroblast cells were seeded in 96-well plates at a concentration of 104 cells per well in 100 µL of DMEM medium containing 10% FBS and 1% penicillin-streptomycin. The cells were then incubated overnight at 37 • C under a controlled atmosphere (5% CO 2 and 80% H 2 O). Next, the culture medium was replaced by the same medium (100 µL) but containing variable concentrations of the part-CaF 2 sample (0-3000 µg·mL −1 ). After 48 h of incubation, the wells were filled with 20 µL cell culture medium (MTS), and then incubated for another 4 h at 37 • C. UV absorbance was measured at 490 nm with a microplate reader (Varioskan Flash, Thermo Scientific, Waltham, MA, USA). Experiments were carried out in 6 replicates and expressed as a percentage of viable cells compared to the control group.

Maximum Tolerated Dose (MTD)
All animals were obtained from the Federal University of Rio de Janeiro Facility and housed under specific pathogen-free conditions. Healthy male Swiss mice (n = 20) aged between 8 and 10 weeks were kept in cages (3 per cage) at a controlled temperature (24-25 • C), and received food and water ad libitum. All experiments were conducted according to the IPEN Animal Ethics Committee approvals (Protocols IPEN-182/2018) and Guidance on Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Market Authorization for Pharmaceuticals (ICH-M3(R2)/2009).
The maximum tolerated dose (MTD) of part-CaF 2 that did not induce unacceptable side effects or toxicity over a specific period of time was determined by 2 methodologies based on both weight loss and clinical signs [51]. For the first methodology, 10 mice were dosed daily. The initial dose for each animal started at 1 mg·kg −1 and all the doses were staggered until reaching 33.79 mg·kg −1 , following the system: day 1 dose = 1 mg·kg −1 ; day 2 dose = day 1 dose + 50%, and so on until day 10, with a final dose of 33.79 mg·kg −1 . For the second methodology, the other 10 mice were dosed based on the ICH M3-R2. The initial dose for each animal started at 1 mg·kg −1 until the maximum dose of 10 mg·kg −1 was reached on day 6. In the last 4 days, all the mice received a fixed dose of 10 mg·kg −1 . The clinical signs were scored based on observation of: (i) general appearance and (ii) body condition. The score was based on the following punctuation: 0 = normal; 1 = slight deviation from normal; and 2 = moderate deviation from normal, as established in Table 1.

Ultrasound and X-ray Imaging Using Part-CaF 2
The absorption of ultrasounds (US) by the part-CaF 2 sample was determined at the Mouse Clinical Institute (Clemson University, Clemson, SC, USA) using a preclinical Vevo 2100 echographie and computer (Visualsonics, Toronto, ON, Canada). The parameters employed were as follows-frequency: 21 MHz; power: 100%; acquisition depth and width of 16.00 mm and 23.04 mm, respectively.

Conclusions and Perspectives
Here, we described the facile production of calcium fluoride particles that are highly crystalline and pure according to XRD and XPS analyses. Compared to other bimodal imaging particles, our results support that part-CaF 2 could offer some tremendous advantages, such as ease of synthesis, low cost, and biodegradability. UV-Vis analysis revealed that part-CaF 2 possesses no absorbance in the visible region. Additionally, XPS confirmed the chemical composition and formula of the material, while the XRD pattern determined its characteristics in terms of the lattice system, cell parameters, and index of crystallinity. Importantly, both in vitro and in vivo experiments using part-CaF 2 did not show any toxicity. Lastly, X-ray and US attenuation tests demonstrated that part-CaF 2 could be used as a bimodal bioimaging contrast agent. Overall, our findings demonstrated that the part-CaF 2 sample holds a great promise for biomedical imaging and opens future potential clinical bioimaging applications. Future work will aim at controlling the size of the particles while maintaining the bimodal contrast signal, implementing the right surface coating, and translating our findings into animal models to understand the biodistribution and pharmacokinetics of this biomaterial and assess its dual bioimaging efficiency in living animals.