1. Introduction

Y₂O₃ is considered to be one of the best hosts for rare-earth (RE) ions for application to cathodoluminescent (CL) displays, catalysis, and luminophors due to its high chemical durability and thermal stability [1, 2]. RE-doped Y₂O₃ phosphors are also promising candidates for biophotonics applications. Due to the similarity of the ionic radius of yttrium ion to that of the other RE ions, RE ions are easily co-doped in host Y₂O₃, and the emission wavelength can be selected like other bioimaging probes such as fluorescent proteins, nanodiamonds, and quantum dots. In addition to the selectivity of the emission wavelength, the excitation wavelength of RE doped Y₂O₃ phosphors can also be changed. Co-doping with Yb ions, whose absorption band is in the 900–1000 nm near-infrared (NIR) wavelength region, enables conversion of lower-energy near-infrared light to higher-energy visible light. Generally, observation of deep regions in specimens is difficult with fluorescence microscopy due to the use of UV-visible light which causes scattering/absorption in biological samples. In addition to the modality for CL observation, Y₂O₃ phosphors have the possibility of allowing deep observation in biological samples by excitation with NIR light [3, 4].

We previously investigated the CL and upconversion luminescence (UPL) properties of Yb-doped Y₂O₃ phosphor particles [5]. Y₂O₃:Tm, Yb phosphor exhibited luminescence via excitation with an electron beam and 980 nm NIR light. By exploiting the multimodal-luminescence properties of Y₂O₃ phosphors, bioimaging will be expanded to a new hybrid imaging technique that combines cathodoluminescence (CL) microscopy and NIR microscopy. Compared with conventional fluorescent imaging, higher-spatial-resolution observation can be expected by CL microscopy due to the use of electron beam excitation. Moreover, CL microscopy has the advantage of enabling color imaging, and various kinds of targets can be recognized by their CL color [6]. In addition, using the NIR wavelength region for both excitation and emission light enables deep observation in biological samples. To date, there have been reports of Y₂O₃:Er, Yb phosphors used as NIR-VIS and NIR imaging probes [7, 8]. Using two kinds of Y₂O₃ phosphors as imaging probes will expand the range of biological applications, with the possibility of exploring cellular functions, from in vivo/deep observation to analysis of specific kinds of target molecules at the nanoscale.

To achieve correlative UPL-CL imaging with Y₂O₃-based phosphors, there are two fundamental problems. First, the phosphor size should be small enough to visualize the distributions of target molecules in CL imaging. Since the spatial resolution of CL microscopy is determined by the spot size of focused electron beam, Y₂O₃ phosphor should be smaller than or equivalent to the size of protein molecules. Second, the luminescence intensities of RE phosphors decrease when the particle size becomes small because luminescence intensities of RE phosphors are proportional to the particle volume [9]. To maintain the detectable luminescence intensity, RE phosphors should be synthesized with optimal concentrations of co-dopants, resulting in a significant influence on the luminescent properties of RE phosphors.

The purpose of the work described in this paper is the development of a new synthesis method for RE phosphors to attain the control of particle size and co-dopants concentrations for correlative UPL-CL imaging. We demonstrated synthesis of Y₂O₃:Tm, Yb and Y₂O₃:Er, Yb NPs with control of the particle size and the concentrations of co-dopants of NPs. To date, numerous studies have proposed advanced synthesis techniques for designing RE oxide-based nanoparticles with sizes of less than 100 nm, including the sol-gel method [10, 11], the laser ablation method [12, 13], the precipitation method [14–18], the solvo-thermal method [19, 20], and the polymerized complex method [21]. We employed a urea-based homogeneous precipitation method, which is a known method of producing uniform-sized RE oxide particles. In this synthesis method, we control not only the particle size but the co-dopants concentrations of RE phosphors. The results of single NP imaging by scanning transmission STEM-CL microscopy are shown, confirming the multimodality of the NPs. Cellular UPL imaging with the NPs was performed under 980 nm NIR light excitation by NIR microscopy.

2. Experimental

2.1. Synthesis of Y₂O₃ phosphors

Yttrium nitrate (Y(NO₃)₃･6H₂O (99.9% purity)), thulium nitrate (Tm(NO₃)₃･xH₂O (99.9% purity)), and ytterbium nitrate (Yb(NO₃)₃･xH₂O (99.9% purity)) were purchased from Kojundo Chemical Laboratory Co., Ltd. Erbium nitrate (Er (NO₃)₃･5H₂O (99.9% purity)) was purchased from Sigma-Aldrich. Urea (for Biochemistry) and ethanol (JIS Special Grade) were purchased from Wako Pure Chemical Industries, Ltd. L-glutamic acid (99.9% purity) was purchased from Kishida Chemical Co. Ltd. All chemicals were used without further purification.

RE oxides Y₂O₃:Tm, Y₂O₃:Tm, Yb and Y₂O₃:Er, Yb were synthesized based on the method used in our previous studies [5, 13]. To synthesize Y₂O₃ NPs, the urea-based homogeneous precipitation method was applied. First, 160 μmol of RE nitrates dissolved in 40 mL of deionized water (DIW) was poured into a 50 mL egg-plant flask and then urea was added to the solution. . Second, the solution was heated to 80 °C while stirring for 1 hour to obtain precursors of NPs. The precursors were separated from residual reactants by ultracentrifugation. To avoid aggregation, the precursors were dispersed in ethanol and were poured into an alumina melting pot and dried at 110 °C. Finally, Y₂O₃ NPs were obtained by calcination at 900 °C for 3 h.

Phosphor pellets were obtained from Y₂O₃:Tm, Yb and Y₂O₃:Er, Yb powders synthesized by the sol-gel method with glutamic acid. The same starting solution of RE nitrates for NPs synthesis was used for powder synthesis. First, 900 μmol of RE nitrates dissolved in 15 mL of DIW was poured into an alumina melting pot with the same molar quantity of L-glutamic acid and stirred at 80 °C for 1 hour. After that, the obtained wet gel was dried at 110 °C for 3 hours. Y₂O₃ phosphor powder was obtained by calcination at 900 °C for 3 h. The phosphor powders were compressed to phosphor pellets 13 mm in diameter with a hydraulic press (Evacuable KBr Die Kit, PIKE technologies) at 10 MPa for 2 minutes.

2.2. Characterization

Particles were observed with transmission electron microscopy (TEM, H-7650, Hitachi). The sizes of the particles were determined from TEM images of more than five hundred particles using the “analyze particles” function of the image-J software (National Institutes of Health). The total masses of RE elements in the precursors were determined with an inductively coupled plasma atomic emission spectroscope (ICP-AES, ISPS-8100, Shimadzu). All precursors were dissolved in 3.5 wt% hydrochloric acid, and the concentrations of RE elements in the solution were determined by calibration of the emission intensities between the precursors and the reference solution at 371.029 nm of yttrium, 313.126 nm of thulium, 337.275 nm of erbium, and 328.937 nm of ytterbium, respectively. Chemical yields of synthesis were derived from the ratio of the total mass of yttrium and thulium in the precursors to that in the starting solution of Y₂O₃:Tm 0.65 mol% NPs. The molar ratio of co-dopant thulium in the precursors was determined by comparison with the total mass of yttrium and thulium in the precursors. Luminescent spectra of Y₂O₃ phosphor pellets under 980 nm NIR light excitation were measured with spectrometers (USB4000 and NIRQUEST512, Oceanoptics), as shown in Fig. 7 (see Appendix).

2.3. Cathodoluminescence imaging of single Y₂O₃:Tm, Yb and Y₂O₃:Er, Yb nanophosphors

CL images of Y₂O₃ NPs were obtained by using a scanning transmission electron microscopy (STEM)-CL system [22–24]. Y₂O₃:Tm 0.21 mol%, Yb 2.16 mol% NPs and Y₂O₃:Er 3.22 mol%, Yb 22.03 mol% NPs were synthesized with 640 mg/mL of urea by the precipitation method described above. The NPs were dispersed on carbon-membrane Cu grids (ELS-C10, UHR-C10, STEM). CL was collected by a parabolic mirror in the STEM (JSM-2100F, JEOL) and was detected by a photomultiplier tube and a photon counting unit. To select the CL of the NPs with the emission wavelength, 465 nm and 660 nm bandpass filters (FF01-465/30-25, FF01-660/52-25, Semrock) were set in front of the photomultipler tube for acquiring the CL from the Y₂O₃:Tm, Yb NPs and the Y₂O₃:Er, Yb NPs, respectively. The electron acceleration voltage was 80 kV.

2.4. Cellular upconversion luminescence imaging using Y₂O₃:Tm, Yb and Y₂O₃:Er, Yb NPs

HeLa cells were cultured in Dulbecco’s modified medium with 10% fetal bovine serum and 1% antibiotic at 37 °C in 5% CO₂. Before being introduced into cells, the Y₂O₃ NPs were dispersed in distilled water and were sterilized by exposing them to UV light for 24 hours. The NPs were dispersed in the medium. One day after passage, the cells were introduced the NPs by an endocytosis reaction. After 24 h from the introduction of the NPs, the cells were rinsed with phosphate buffered saline (PBS (-), Wako) three times and were fixed with 4% paraformaldehyde in PBS (-). After fixation, the cells were observed with a modified laser scanning microscope (C.1, Nikon). Luminescent images were constructed by raster scanning the beam from a 980 nm NIR diode laser (IRM980TR-500, Laser Century) using the scanning system of the microscope without pinhole. For acquisition of UPL from the NPs, an objective lens (LR Plan Apo NIR 20x, NA 0.40, Nikon), a short-pass filter (FF01-950/SP-25, Semrock), band-pass filters (Hard Coated Bandpass Filter 800 nm 25 mm, OD4, Edmund Optics for acquisition of UPL from Y₂O₃: Tm, Yb NPs, FF01-660/52-25, Semrock for acquisition of UPL from Y₂O₃: Er, Yb NPs), and a photomultiplier tube (H7844, Hamamatsu Photonics) mounted between the objective and the scanner were used.

3. Results and discussion

3.1.Evaluation of particle properties

We investigated the effect of the precipitant urea concentration in the precipitation reaction on the size of the nanoparticles. Figures 1(a)-1(d) and 1(e) show TEM images and the particle size distribution of precursors synthesized with various concentrations of urea. In the case of lower concentrations of urea, the particle sizes were large and were widely distributed. Precursors synthesized with 40 mg/mL of urea had a wide distribution, from tens of nanometers to 500 nm. When the concentration of urea was increased to 80 mg/mL, the size distribution was narrowed; however, the particle size converged around 350-500 nm. In contrast, a further increase of urea concentration caused a decrease in particle size and narrowing of the size distribution. The sizes of the precursors synthesized with 320 mg/mL of urea were in the ranges 160–200 nm and 40–60 nm. The sizes and size distributions of the precursors continued to decrease as the concentration of urea was increased, and particle sizes converged around 40-80 nm. Figure 1(f) shows the relationship between the mean diameter of nanoparticles and the concentration of urea. With a lower amount of urea, precursors had large size, at greater than 100 nm, with a wide size distribution. Precursors with particle sizes of 143.0 ± 43.0 nm, 189.0 ± 79.7 nm, and 269.1 ± 140.4 nm were obtained by using 5, 10, and 40 mg/mL of urea, respectively. An increase of the mean particle size was observed as the concentration of urea increased, and a maximum particle size of around 409.8 ± 61.2 nm was observed with 80 mg/mL of urea. On the other hand, the mean size and size distributions of particles decreased with higher concentrations of urea. Finally, the particle size converged to 42.3 ± 11.8 nm with 720 mg/mL of urea.

 

Fig. 1 (a-d) Transmission electron microscopy images of precursors synthesized with 40, 80, 320, and 640 mg/mL of urea. Scale bar: 1 μm. (e) Size distributions and (f) average diameter of precursors synthesized with various concentrations of urea. Error bars in (f) serve as standard deviations of particle sizes.

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Figure 2 shows the chemical yields of synthesized precursors with various amounts of urea. The yield is defined as the weight ratio of RE elements (yttrium and thulium) between in the synthesized precursor and the starting solution determined by ICP-AES. The chemical yield of synthesis in each condition was calculated from three samples. When the total RE weight in the starting solution was defined as 100%, the yields of precursors were 68.5 ± 5.7% and 84.1 ± 3.3% with 40 mg/mL and 80 mg/mL of urea, respectively. An increase in the urea concentration provided an increase in the yield of precursors, and yields of higher than 90% were attained with 160, 320, 640, and 720 mg/mL urea.

 

Fig. 2 Chemical yields of synthesis with various concentrations of urea determined by comparison of total mass of yttrium and thulium in the synthesized precursors and in the starting solution.

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We consider that the decrease in particle size in the reaction with excessive urea was derived from rapid nuclei formation in the initial stage of the reaction. Briefly, the precipitation reaction includes three reactions: (1) nuclei formation at the critical point of supersaturation, (2) particle growth, and (3) unification of particles. Use of a large amount of urea causes a large amount of nuclei formation and consumption of reactants. As a result, particle growth is suppressed due to the lack of reactants in the solution. The high yields and the small sizes of precursors with high-concentration urea support this hypothesis. Experimentally, the use of much higher concentrations of urea still decreased the mean particle size of precursors, but the precursors were agglomerated. It may be necessary to suppress the unification of particles by using a surfactant to obtain smaller precursors with dispersed conditions when a very high concentration of urea is applied.

3.2. Relationship between luminescent intensity and concentrations of co-dopants in Y₂O₃ phosphors

We evaluated the relation between the RE concentration and upconversion luminescence intensity. To obtain accurate RE concentrations, phosphors powders were synthesized by the sol-gel method, whose final product has a composition corresponding to that of the starting materials. The phosphor powders were pressed into pellet shapes. Luminescence intensities of RE phosphor pellets were evaluated by changing the concentrations of co-dopants. Figure 3 shows the UPL spectra of Y₂O₃:Tm, Yb phosphor pellets synthesized with various concentrations of co-dopants under 980 nm NIR light excitation at a power of 200 mW. Y₂O₃:Tm, Yb phosphors exhibited UPL around 810 nm attributed to the ¹G₄ → ³H₅ and ³H₄ → ³H₆ energy transitions of Tm³⁺. To understand the influence of co-dopant Tm concentrations on the luminescence intensity, UPL spectra of phosphors were obtained with three combinations of co-dopant concentrations; Y₂O₃:Tm 0.12 mol%, Yb 2.04 mol%, Y₂O₃:Tm 0.23 mol%, Yb 2.04 mol%, and Y₂O₃:Tm 0.58 mol%, Yb 1.98 mol%. The molar ratio of the activator Tm³⁺ was varied in the range 0.1–0.6 mol%. The concentration of the sensitizer Yb³⁺ co-dopant was fixed at around 2 mol%. The maximum luminescent intensities in the spectra were normalized to the peak intensities and shown as a bar chart. The phosphors showed an increase in luminescence as the concentration of the activator Tm³⁺ increased, and the brightest luminescence was observed with 0.23 mol% of Tm. On the contrary, co-doping with 0.58 mol% of Tm caused quenching of the luminescence. The activator Tm³⁺ does not absorb 980 nm light directly and can emit luminescence via upconversion energy transfer from the sensitizer Yb³⁺ whose absorption band is 900–1000 nm. Co-doping with high concentrations of Tm easily caused quenching of the UPL. The results indicated that the luminescent intensities of phosphors are influenced even by a 0.1 mol% difference in concentrations of the co-dopants. To distribute bright NPs, precise control of co-dopant concentrations in the synthesis of NPs is highly desired.

 

Fig. 3 Comparison of the upconversion luminescence spectra and peak intensities, around 750–850 nm, of Y₂O₃: Tm, Yb phosphor pellets synthesized with various molar ratios of Tm³⁺ co-dopants.

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3.3. Control of co-dopant molar ratio in nanophosphors

The molar ratio of the co-dopant Tm in precursors of the NPs was investigated. Figure 4 shows a comparison value of Tm concentrations between precursors and the starting solution. Each molar ratio was determined from three samples by ICP-AES elemental analysis. All precursors were synthesized from a starting solution that included 0.65 mol% of co-dopant Tm. When 0.65 mol% of co-dopant thulium in the starting solution was defined as 100%, precursors included higher molar ratios of Tm in precursors, namely, 269.6 ± 16.9%, 270.3 ± 5.0%, and 124.7 ± 6.1% with 5, 10, and 40 mg/mL of urea, respectively. Use of a larger amount of urea resulted in precursors that included almost equal molar ratios of the co-dopant thulium in the starting solution, namely, 99.0 ± 4.6%, 94.7 ± 1.1%, and 97.0 ± 1.0% with 80, 320, and 640 mg/mL of urea, respectively.

 

Fig. 4 Molar concentration ratio of co-dopant Tm between precursors and starting solution.

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The mechanism of co-dopant precipitation in the precursors can be explained by nuclei formation. Nuclei of RE hydroxides are generated under super saturation whose critical point is controlled with the reaction temperature, the amount of RE ions, and the pH value of the solution. Concentrations of RE elements in the starting solution play a dominant role in determining the critical point of super saturation, and nuclei formation of each RE hydroxides starts with different solution pH. RE cations (Re³⁺) form hydroxides, as shown in the following reaction:

In addition to control the particle size, use of excessive urea was found to be effective also for control of the co-dopant concentrations of NPs. Since nuclei formation of co-dopant thulium hydroxides occurred prior to that of the host yttrium hydroxides during reactions with less than 40 mg/mL of urea, use of excessive urea allowed an increase of solution pH in an initial stage of the precipitation reaction (Fig. 9), and the host and co-dopant ions were consumed simultaneously to form precursors.

3.4. Cathodoluminescence imaging of Y₂O₃:Tm, Yb and Y₂O₃:Tm, Yb nanophosphors

We demonstrated CL imaging of single Y₂O₃:Tm 0.21 mol%, Yb 2.16 mol% and Y₂O₃:Er 3.22 mol%, Yb 22.03 mol% NPs using the STEM-CL system. Figure 5(a) shows STEM-CL images of Y₂O₃:Tm, Yb NPs. The NPs used for cellular UPL imaging were observed in STEM-CL imaging. The wavelength region around 450–480 nm was employed for CL imaging of the Y₂O₃:Tm, Yb NPs. Distributions of two 40–50 nm-sized NPs were visualized in the CL image, and the positions of the NPs corresponded to those in the STEM image. After imaging, the stability of the NPs to exposure with 80 kV-accelerated electrons was investigated, as shown in Fig. 5(b). The electron beam was focused at the center position of the nanoparticle located to upper region in Fig. 5(a). The NP kept emitting intense blue CL for 60s. High stability of NPs was also confirmed in CL imaging of Y₂O₃:Er, Yb NPs. In Fig. 5(c), three Y₂O₃:Er, Yb NPs were recognized by red CL at 640–680 nm. The intensity of CL depended on the size of NPs: a large NP positioned at the left part, indicated by the character alpha, in the CL image emitted stronger red CL compared with those of NPs indicated by characters beta and gamma at the right part. Meanwhile, every NP showed high CL stability under electron beam exposure, and even 40 nm-sized NPs maintained CL emission for 60 s. The above results indicate that highly emissive and stable NPs were synthesized with excessive urea.

 

Fig. 5 Cathodoluminescence images (a, c) and stabilities of cathodoluminescence intensity (b, d) of Y₂O₃: Tm, Yb (a, b) and Y₂O₃: Er, Yb (c, d) nanophosphors. Excitation source: electron beam. Acquisition wavelength: 450-500 nm (a, b), 640-680 nm (c, d); Acceleration voltage: 80 kV; Image size: 240 nm x 240 nm; pixel size: 2 nm (b), 2.5 nm (d); Scanning speed: 10 ms/pixel (a), 5 ms/pixel (c).

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3.5. Cellular upconversion luminescence imaging using Y₂O₃:Tm, Yb and Y₂O₃:Er, Yb nanophosphors

We demonstrated UPL imaging of HeLa cells containing Y₂O₃:Tm 0.21 mol%, Yb 2.16 mol% and Y₂O₃:Er 3.22 mol%, Yb 22.03 mol% NPs, as shown in Fig. 6. To image distributions of NPs, we used UPL around 770–830 nm, attributed to the ¹G₄ → ³H₅ and ³H₄ → ³H₆ energy transitions of Tm³⁺ [6], and UPL around 640–680 nm, attributed to ⁴F_9/2 → ⁴I_15/2 energy transitions of Er³⁺ [7, 8]. The UPL from the two kinds of NPs was clearly recognized as luminescent spots in HeLa cells under 980 nm NIR light excitation. Since both excitation and emission wavelengths are in the range of 650 to 1000 nm, which have low absorption and scattering coefficients in the body [28], these NPs are advantageous for observing deeper regions in biological samples.

 

Fig. 6 Cellular bright-field (a, c) and upconversion luminescence (b, d) images of HeLa cells using Y₂O₃:Tm, Yb (a, b) and Y₂O₃:Er, Yb (c, d) nanophosphors. Excitation wavelength: 980 nm; Intensity of 980 nm near-infrared laser light: 14.5 mW (b), 9.7 mW (d); Acquisition wavelength: 770-830 nm (b), 640-680 nm (d); Scanning speed 61.44 µs/pixel.

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The demonstrated results indicate the possibility of advanced multi-colored bioimaging with CL microscopy and NIR microscopy. Y₂O₃ NPs can be made to have a tunable emission wavelength region under both electron beam and 980 nm NIR light excitation by changing the co-dopant elements, and it is possible to discriminate the kinds of targets by the luminescence color at a glance. Moreover, the NPs have high stability to electron beam exposure, and no bleaching of NPs was observed. The multimodality, color selectability, and high stability of the NPs will expand the conventional correlative bioimaging to consecutive CL microscopy and NIR microscopy observation.

4. Conclusion

In conclusion, Y₂O₃:Tm, Yb and Y₂O₃: Er, Yb NPs were successfully designed as multimodal UPL/CL imaging probes. Control of both particle size and concentrations of co-dopant of NPs were attained by proper use of precipitant urea. Synthesis of precursors with size control less than 50 nm and chemical yield greater than 90% were achieved by homogeneous precipitation method. Relationship between luminescent properties and concentrations of co-dopants was investigated. It was found that use of excessive urea enabled to obtain precursors with precise control of co-dopant concentrations. Multimodalities of the NPs were demonstrated by CL microscopy and NIR microscopy observation. The developed NPs will expand new correlative bioimaging applications with CLEM technique including CL and NIR microscopies, as wide scaled correlative observations from molecular distributions to deep tissue observation.

Appendix

Figure 7 shows the optical setup to obtain luminescent spectra of Y₂O₃ phosphor pellets. Near-infrared 980 nm laser light from infrared diode laser source (IRM980TR-500, Laser Century) was lead to phosphor pellets through pair of achromatic lens (AC-254-075-C, Thorlabs) as the beam expander (BE) and silver coated mirror (M). Near-infrared laser light was focused to phosphor pellet by achromatic lens (AC-254-075-C, Thorlabs; L1) and filtered by short-pass filter (BlightLine, fluorescence filter 950/SP, Semrock; F1) or long-pass filter (RazorEdge LongPass 980, Semrock; F2). Upconversion luminescence and NIRL from phosphor pellet were collected by silver-coated parabolic mirror (MPD254254-90-P01, Thorlabs; PB) and were lead to spectroscopies (USB4000, Oceanoptics, NIRQUEST, NQ51A0577, Oceanoptics) through a two-branched optical fiber (CUSTOM-BIF-6174546, 1000 um VIS/NIR, Oceanoptics). Obtained spectral information was fixed by calibration light source device (HL2000, Oceanoptics).

 

Fig. 7 Optical setup for the acquisition of luminescence from phosphor pellets.

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Figure 8 shows theoretical pH value of solution on a critical point of precipitation reaction. RE ions such as yttrium, thulium, erbium, and ytterbium ions (Re³⁺) form hydroxides as shown in below reaction:

(3)$${\mathrm{Re}}^{3+}+3O{H}^{-}\rightleftarrows \mathrm{Re}{(OH)}_3.$$

The pH value of super saturation is calculated using the following equation:

(4)$$PH=14+{\log }_{10}\left[O{H}^{-}\right]=14+{\log }_{10}{\left(\frac{k_{sp}}{\left[{\mathrm{Re}}^{3+}\right]}\right)}^{\frac{1}{3}},$$

where K_sp is the solubility product of each RE-earth hydroxides [25–27].

 

Fig. 8 Maximum and minimum predicted pH value of super saturation of nuclei formation of rare-earth hydroxides from (a) (Y_0.978Tm_0.002Yb_0.02)(NO₃)₃ and Y_0.9765Er_0.008Yb_0.0155(NO₃)₃ start solution.

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Figure 9 shows pH value of the solution in precipitation reaction with various concentrations of urea.160 μmol of rare-earth nitrates (158.96 μmol of yttrium nitrate and 1.04 μmol of thulium nitrate) was dissolved into DIW of 40 mL to prepare start solution of Y₂O₃: Tm 0.65 mol%. The start solution was heated to 80 °C for 1h to obtain precursors described above. pH value of solution was measured every 30 seconds using a pH meter (F-52 and standard pH sensor 9615S-10D, HORIBA).

 

Fig. 9 pH value of solution during the precipitation reaction with various concentrations of urea.

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Acknowledgments

This research was supported by a Grant-in-Aid from Japan Society for the Promotion of Science(JSPS), Ministry of Education, Culture Sports, Science and Technology (MEXT) Photonics Advanced Research Center Program (Osaka University), a grant from the Kazato Research Foundation for “Kazato Research Encouragement Prize 2015”, Tokyo, Japan, and a Grant-in-Aid for Scientific Research on Innovative Area “Nanomedicine Molecular Science” (No. 2306) from MEXT, Japan.

References and links

1. W. M. Yen, S. Shionoya, and H. Yamamoto, Phosphor Handbook (CRC Press, 2006)

2. D. Matsuura, “Red, green, and blue upconversion luminescence of trivalent-rare-earth ion-doped Y₂O₃ nanocrystals,” Appl. Phys. Lett. 81(24), 4526–4528 (2002). [CrossRef]  

3. D. K. Chatterjee, A. J. Rufaihah, and Y. Zhang, “Upconversion fluorescence imaging of cells and small animals using lanthanide doped nanocrystals,” Biomaterials 29(7), 937–943 (2008). [CrossRef]   [PubMed]  

4. L. Cheng, K. Yang, S. Zhang, M. Shao, S. Lee, and Z. Liu, “Highly-Sensitive Multiplexed in vivo Imaging Using PEGylated Upconversion Nanoparticles,” Nano Res. 3(10), 722–732 (2010). [CrossRef]  

5. S. Fukushima, T. Furukawa, H. Niioka, M. Ichimiya, J. Miyake, M. Ashida, T. Araki, and M. Hashimoto, “Y₂O₃:Tm,Yb nanophosphors for correlative upconversion luminescence and cathodoluminescence imaging,” Micron 67, 90–95 (2014). [CrossRef]   [PubMed]  

6. H. Niioka, T. Furukawa, M. Ichimiya, M. Ashida, T. Araki, and M. Hashimoto, “Multicolor cathodoluminescence microscopy for biological imaging with nanophosphors,” Appl. Phys. Express 4(11), 112402 (2011). [CrossRef]  

7. F. Vetrone, J. C. Boyer, J. A. Capobianco, A. Speghini, and M. Bettinelli, “Significance of Yb³⁺ concentration on the upconversion mechanisms in codoped Y₂O₃: Er³⁺, Yb³⁺ nanocrystals,” J. Appl. Phys. 96(1), 661–667 (2004). [CrossRef]  

8. E. Hemmer, H. Takeshita, T. Yamano, T. Fujiki, Y. Kohl, K. Löw, N. Venkatachalam, H. Hyodo, H. Kishimoto, and K. Soga, “In Vitro and In Vivo investigations of upconversion and NIR emitting Gd₂O₃:Er³⁺,Yb³⁺ nanostructures for biomedical applications,” J. Mater. Sci. Mater. Med. 23(10), 2399–2412 (2012). [CrossRef]   [PubMed]  

9. E. Hemmer, N. Venkatachalam, H. Hyodo, A. Hattori, Y. Ebina, H. Kishimoto, and K. Soga, “Upconverting and NIR emitting rare earth based nanostructures for NIR-bioimaging,” Nanoscale 5(23), 11339–11361 (2013). [CrossRef]   [PubMed]  

10. S. Y. Lee and R. S. Feigelson, “c-axis lithium niobate thin film growth on silicon using solid-source metalorganic chemical vapor deposition,” J. Mater. Res. 14(06), 2662–2667 (1999). [CrossRef]  

11. D. Dong, S. Jiang, Y. Men, X. Ji, and B. Jiang, “Nanostructured hybrid organic-inorganic lanthanide complex films produced in situ via a sol-gel approach,” Adv. Mater. 12(9), 646–649 (2000). [CrossRef]  

12. Y. Onodera, T. Nunokawa, O. Odawara, and H. Wada, “Upconversion properties of Y₂O₃:Er, Yb nanoparticles in prepared by laser ablation in water,” J. Lumin. 137, 220–224 (2013). [CrossRef]  

13. T. Furukawa, H. Niioka, M. Ichimiya, T. Nagata, M. Ashida, T. Araki, and M. Hashimoto, “High-resolution microscopy for biological specimens via cathodoluminescence of Eu- and Zn-doped Y₂O₃ nanophosphors,” Opt. Express 21(22), 25655–25663 (2013). [CrossRef]   [PubMed]  

14. H. H. Willard and N. K. Tang, “A study of the precipitation of aluminum basic sulfate by urea,” J. Am. Chem. Soc. 59(7), 1190–1196 (1937). [CrossRef]  

15. T. Ishikawa and E. Matijević, “Preparation and Properties of Uniform Colloidal Metal Phosphates,” J. Colloid Interface Sci. 123(1), 122–128 (1988). [CrossRef]  

16. B. Aiken, W. P. Hsu, and E. Matijević, “Preparation and properties of monodispersed colloidal particles of lanthanide compounds: III, yttrium(III) and mixed yttrium(III)/cerium(III) systems,” J. Am. Ceram. Soc. 71(10), 845–853 (1988). [CrossRef]  

17. S. Sohn, Y. Kwon, Y. Kim, and D. Kim, “Synthesis and characterization of near-monodisperse yttria particles by homogeneous precipitation method,” Powder Technol. 142(2-3), 136–153 (2004). [CrossRef]  

18. J. G. Li, T. Ikegami, J. H. Lee, and T. Mori, “Well-sinterable Y₃Al₅O₁₂ powder from carbonate precursor,” J. Mater. Res. 15(7), 1514–1523 (2000). [CrossRef]  

19. X. C. Jiang, C. H. Yan, L. D. Sun, A. G. Wei, and C. S. Liao, “Hydrothermal homogeneous urea precipitation of hexagonal YBO₃:Eu³⁺ nanocrystals with improved luminescent properties,” J. Solid State Chem. 175(2), 245–251 (2003). [CrossRef]  

20. J. Zhang and J. Lin, “Vaterite-type YBO₃:Eu³⁺ crystals: hyrothermal synthesis, morphology and photoluminescence properties,” J. Cryst. Growth 271(1-2), 207–215 (2004). [CrossRef]  

21. M. Kakihana, “Synthesis of high-performance ceramics based on polymerizable complex method,” J. Ceram. Soc. Jpn. 117(1368), 857–862 (2009). [CrossRef]  

22. N. Yamamoto, K. Araya, and F. J. García de Abajo, “Photon emission from silver particles induced by a high-energy electron beam,” J. Phys. Rev. B 64(20), 205419 (2001). [CrossRef]  

23. N. Yamamoto, S. Ohtani, and F. J. García de Abajo, “Gap and Mie plasmons in individual silver nanospheres near a silver surface,” Nano Lett. 11(1), 91–95 (2011). [CrossRef]   [PubMed]  

24. T. Furukawa, S. Fukushima, H. Niioka, N. Yamamoto, J. Miyake, T. Araki, and M. Hashimoto, “Rare-earth-doped nanophosphors for multicolor cathodoluminescence nanobioimaging using scanning transmission electron microscopy,” J. Biomed. Opt. 20(5), 056007 (2015). [CrossRef]   [PubMed]  

25. T. Moeller and N. Fogel, “Observations on the rare earths. LXI. precipitation of hydrous osides or hydroxides from perchlorate solutions,” J. Am. Chem. Soc. 73(9), 4481 (1951). [CrossRef]  

26. M. Tamres, “Solubility constants of metal oxides, metal hydroxides and metal hydroxide salts in aqueous solution,” Inorg. Chem.3(2), 307 (1964).

27. Z. S. Cetiner, S. A. Wood, and C. H. Gammons, “The aqueous geochemistry of the rare earth elements. Part XIV. The solubility of rare earth element phosphates from 23 to 150°C,” Chem. Geol. 217(1–2), 147–169 (2005). [CrossRef]  

28. A. M. Smith, M. C. Mancini, and S. Nie, “Bioimaging: Second window for in vivo imaging,” Nat. Nanotechnol. 4(11), 710–711 (2009). [CrossRef]   [PubMed]