Photoluminescence, cytotoxicity and in vitro imaging of hexagonal terbium phosphate nanoparticles doped with europium

Abstract

Luminescent TbPO₄ nanoparticles were synthesized via a citric-acid-mediated hydrothermal route. Eu³⁺ doping of TbPO₄ enables an efficient Tb³⁺-to-Eu³⁺ energy transfer, leading to a four-fold increase of the absolute emission quantum yield (QY), compared to that of undoped TbPO₄. To check the potential of biological use, we conducted in vitro biological experiments on human cervical carcinoma HeLa cells incubated with TbPO₄:Eu nanoparticles. TbPO₄:Eu nanoparticles can be successfully internalized into the cells, and they show bright intracellular luminescence and very low cytotoxicity. Photoluminescence intensity dependence upon time demonstrates that Eu³⁺-doped TbPO₄ nanoparticles are highly resistant to photobleaching. Our present work represents a demonstration of the use of rare-earth-based nanocrystals as a biological labeling agent because they combine several advantages including high emission quantum yield, long luminescence lifetime, low cytotoxicity and high photostability.

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1. Introduction

There is an increasing interest in the development of luminescent labeling materials for bioimaging.^1–5 Conventional luminescent labels include organic dyes^3,4 and semiconductor quantum dots (QDs) such as CdSe and CdTe.^1,2,5 However, these labeling agents also have some intrinsic limitations in their practical biological uses. For example, the main drawback of organic dyes is photobleaching,⁶ while QDs are still controversial due to their inherent toxicity and chemical instability.⁷

Recently, rare-earth-based nanoparticles have been proposed as a new generation of biological luminescent labels and as viable alternatives to conventional labels,^8–14 due to their attractive chemical, optical and biocompatible features, such as tunable emission colors, high photochemical stability, long luminescence lifetime (up to several ms), low photobleaching and low toxicity.^11,14 Moreover, these rare-earth-based probes also possess additional distinctive features, such as sharp emission bands and large Stokes shifts (as much as several hundred nanometres),¹⁵ leading to an improved sensitivity.

Metal phosphates represent an important family of materials and constitute good host matrixes for luminescent rare earth ions.^16–18 They have been used as active components in a wide range of applications such as high performance luminescent devices, magnets, catalysts, fluorescence labels for biological detection, and other functional materials based on the electronic, optical, and chemical characteristics resulting from the 4f shell of their ions.¹⁹ Very recently, much effort regarding metal phosphate has been devoted to novel synthetic routes and their new potential in applications. Yan's group²⁰ reported on the synthesis of orderly aligned and highly luminescent monodisperse rare-earth orthophosphate nanocrystals by a limited anion-exchange reaction in solution phase. Luo et al.²¹ reported the synthesis of ordered cubic mesoporous yttrium phosphates (YPO₄) via a hard template method and the corresponding photoluminescence properties upon rare earth doping. Our previous work²² demonstrated luminescent CePO₄:Tb nanocrystals as a novel oxygen sensing material based on redox responsive reversible luminescence. Epple's group^14,16 claimed that calcium phosphate is a very promising biomaterial for bioimaging, photodynamic therapy and as DNA carriers due to its high biocompatibility and good biodegradability. Wong's group¹⁰ used a simple and effective template-mediated protocol to synthesize CePO₄:Tb nanowires and demonstrated their potential applications in biological imaging and cellular labeling.

It is well known that nanosized luminescent materials with small particle size show lower luminescence quantum yield than those of materials with larger particle size or the corresponding bulk materials due to a low crystallinity and a number of surface defect states. A high luminescence quantum yield (QY) is required for the use of nanoparticles in biological imaging and labeling. In this work, photoluminescent TbPO₄ nanoparticles were synthesized via a citric-acid-mediated hydrothermal route and investigated as luminescent labeling agents. It is found that the undoped TbPO₄ nanoparticles show a low photoluminescence QY of only 0.07, while Eu³⁺ doping in TbPO₄ matrix enables an efficient Tb³⁺-to-Eu³⁺ energy transfer, leading to a four-fold increase of QY. Such highly luminescent nanoparticles were used to conduct in vitro biological experiments using human cervical carcinoma HeLa cells. The cellular permeability, cytotoxicity, photostability and in vitro imaging of fluorescent TbPO₄:Eu nanoparticles were studied. Our work demonstrated that rare-earth-based luminescent nanomaterials have a high potential for biological imaging and labeling.

2. Results and discussion

2.1 Size and morphology

The as-synthesized product corresponds to hexagonal rhabdophane-type TbPO₄ crystals, as revealed by X-ray diffraction studies (JCPDS No. 20-1244; Fig. S1 in the ESI†). The size and morphology of the product were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations. A SEM image (Fig. 1a) reveals that the sample consists of hexagonal prism nanoparticles. High resolution TEM and the corresponding Fourier transform images provide deep insight into the crystalline size and quality, and preferential growth axis (the insets of Fig. 1c). The products are single-crystalline nanoparticles, ranging from 60 to 80 nm in diameter. They are highly crystalline without visible defects and dislocations. Generally, lanthanide phosphates with hexagonal rhabdophane-type structure prefer to grow along the [001] direction in the absence of any organic additive acting as chelating agent, leading the formation of rod-shaped particles with high aspect ratio.^17,18 In our synthesis, citric acid acts as chelating agent of Tb³⁺. Moreover, citrate ions can also selectively bind to specific crystallographic facets, inhibiting or promoting the growth along specific directions, finally leading to dramatic modifications of the final morphologies of the particles.^17,23 In this case, one of the consequences of the presence of citrate ions is the inhibition of the [001] growth direction and the resulting relative enhancement of the growth sideways along the [100] and [010] directions, which gives rise to highly symmetric hexagonal-prism-shaped nanoparticles with well-defined crystallographic facets. Similar results were observed in the synthesis of other types of inorganic materials.^24,25

Fig. 1 SEM (a) and TEM (b and c) images of TbPO₄ synthesized by a citric-acid-mediated hydrothermal route.

2.2 Photoluminescence

Fig. 2 presents the emission spectra of TbPO₄ without and with Eu³⁺ doping. For undoped TbPO₄ nanoparticles excited at 377 nm, the spectrum displays a series of emission lines ascribed to the Tb³⁺ intra-4f⁸⁵D₄ → ⁷F_6–3 electronic transitions. Upon doping of a small amount of Eu³⁺, we can observe additional emission lines appearing from 585 to 710 nm ascribed to the Eu³⁺ intra-4f⁶⁵D₀ → ⁷F_0–4 transitions. The simultaneous observation of the emission arising from Tb³⁺ and Eu³⁺ excited states suggests the possible existence of Tb³⁺-to-Eu³⁺ energy transfer.

Fig. 2 Room temperature emission spectra of pure (full line) and 0.3 mol% Eu³⁺-doped TbPO₄ (dashed line) excited at 377 nm.

To verify whether the energy transfer of Tb³⁺-to-Eu³⁺ is present, we measured the excitation spectra of TbPO₄ without and with Eu³⁺ doping (Fig. S2 in the ESI†). The excitation of the Eu-doped samples were monitored within the Tb³⁺⁵D₄ → ⁷F₅ (544 nm) and Eu³⁺⁵D₀ → ⁷F₄ (697 nm) transitions, respectively. The former excitation spectra is similar to the one acquired for the undoped TbPO₄ sample. The spectra monitored within the Eu³⁺ emission lines exhibit a series of Tb³⁺ related transitions and a low-relative intensity peak attributed to the Eu³⁺⁷F₀ → ⁵L₆ transition. The observation of the Tb³⁺ excited levels in the excitation spectra monitored within the Eu³⁺ intra-4f⁶ transitions is a clear evidence of the occurrence of Tb³⁺-to-Eu³⁺ energy transfer.

The existence of the energy transfer from Tb³⁺ to Eu³⁺ can be also evidenced by the dependence of the ⁵D₄ lifetime values on the Eu³⁺ concentrations. The luminescence decay of pure TbPO₄ and doped TbPO₄ by monitoring the Tb³⁺ emission at 543 nm were measured. All the curves are well-reproduced by a single exponential function, yielding to lifetime values of 0.495 ± 0.001 ms for the pure TbPO₄ sample and 0.354 ± 0.001, 0.230 ± 0.001 and 0.109 ± 0.001 ms for TbPO₄ doped with 0.001, 0.003 and 0.005 mol% Eu³⁺, respectively. The presence of the acceptor Eu³⁺ increases the transition rate of Tb³⁺ excited states and thus decreases the Tb³⁺⁵D₄ lifetime, proving the existence of Tb³⁺-to-Eu³⁺ energy transfer.

The dynamics of the Eu³⁺ luminescence (monitored at 697 nm, ⁵D₄ → ⁷F₆ transition of Eu³⁺) under pulsed excitation at 355 nm also confirms the occurrence of Tb³⁺-to-Eu³⁺ energy transfer in TbPO₄:Eu sample (Fig. 3). It is observed that the population loading presents a longer rise time following the excitation laser pulse, as marked by a circle in Fig. 3. The slow rise time is due to an increase of population of the Eu³⁺:⁵D₀ state via energy transfer from Tb³⁺. Eu³⁺-doped TbPO₄ shows a luminescence lifetime of Eu³⁺ as long as 2.5 ms.

Fig. 3 Room temperature emission decay curves of TbPO₄:5%Eu³⁺ monitored with Eu³⁺ emission at 697 nm (⁵D₀ → ⁷F₄ transition).

Fig. 4 shows the Eu³⁺ emission intensity of samples as a function of Eu³⁺ concentration. It can be seen that the luminescence of Eu³⁺ increases with Eu³⁺ concentration and reaches a maximum value around 5 mol% Eu³⁺. At this concentration range, the Tb³⁺-to-Eu³⁺ energy transfer is dominant to the luminescence of materials and Tb³⁺ emissions are almost completely quenched. For Eu³⁺ concentration higher than 5%, the emission intensity starts to decrease. The increase of Eu³⁺ concentration above 5% reduces the distance between the luminescent centers and thus increases the probability of nonradiative energy migration between Eu³⁺ ions leading to nonradiative deexcitation, which is known as the concentration quenching.

Fig. 4 Emission intensity of Eu³⁺ as a function of Eu³⁺ concentration.

The photoluminescence features were further quantified through the estimation of the absolute emission QY as a function of the Eu³⁺ concentration. For all the samples, the maximum quantum yield value was attained under excitation at 377 nm. Table 1 records the QY values of undoped TbPO₄ and TbPO₄ doped with various concentrations of Eu³⁺. The undoped TbPO₄ shows a low QY value of only 0.07, while a small amount of Eu³⁺ doping improves the QY significantly, that is, a two-fold increase of QY (0.14) is acquired for TbPO₄:Eu_0.003. The QY value continues to increase with the increase of Eu³⁺ concentration up to 5 mol%, where a maximum QY value of 0.30 was acquired. This is in good agreement with the tendency observed in Fig. 4. The remarkable increase of the QY of samples with Eu³⁺ doping is attributed to an efficient Tb³⁺-to-Eu³⁺ energy transfer. Under direct intra-4f⁶ excitation (⁷F₀ → ⁵L₆, 393 nm) the quantum yield value of the TbPO₄:Eu samples lies below the detection limits of our equipment (0.01), confirming that the preferential path for the Eu³⁺ excitation is via the Tb³⁺ excited levels. Luminescent nanoparticles with high luminescence QY and long excited-state lifetime are preferred for their biological application, as will be demonstrate below.

Table 1 Absolute emission quantum yield (QY) of TbPO₄ doped with various concentration of Eu³⁺

Tb1−xPO4:Eux	x = 0	x = 0.003	x = 0.01	x = 0.03	x = 0.05	x = 0.06
QY	0.07	0.14	0.19	0.26	0.30	0.24

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2.3 Cellular uptake and imaging

Generally, the cellular permeability and cytotoxicity characteristics of fluorescent nanomaterials are critical to their biological applications. To check the possibility of the use of TbPO₄:Eu nanoparticles as luminescent biological labels, we conducted in vitro biological experiments using human cervical carcinoma HeLa cells. We choose TbPO₄ doped with 5 mol% Eu³⁺ for all biological experiment since it shows the highest luminescence QY. As a control experiment, HeLa cells alone show almost no background fluorescence (Fig. 5a). Nonetheless, upon incubation, incorporation of TbPO₄:Eu nanoparticles into HeLa cells was confirmed by a fluorescence microscopy. Fig. 5b and c show fluorescence and bright-field optical microscopy images of HeLa cells after incubation with 0.1 mg mL⁻¹ of TbPO₄:Eu nanoparticles for 8 h. As observed, the nanoparticles can permeate into the cell membrane and display bright intracellular luminescence from Eu³⁺ (Fig. 5b). These inorganic phosphate nanoparticles clearly retained their intrinsic fluorescent properties upon cellular internalization. Moreover, the corresponding bright-field measurements taken after incubation with the nanoparticles confirmed that the cells were viable throughout the imaging experiments (Fig. 5c) and that there were no evident regions of cell death. To check the spatial localization of foreign materials within the cells, we took a series of Z-stack images of the cell (e.g., top to bottom) at 1 µm “slice” intervals of the HeLa cells stained with TbPO₄:Eu nanoparticles. A representative fluorescence image (Fig. 5d) taken from an internal slice clearly shows a red fluorescence within the cells. This indicates that TbPO₄:Eu nanoparticles are localized within the interior environment of the HeLa cells. Very recently, Wong's group¹⁰ and our previous work²⁶ have obtained similar results for CePO₄:Tb nanomaterials with green luminescence, which also shows intense intracellular luminescence after incubated with HeLa cells. This indicates the high potential of rare-earth-based luminescent materials toward applications in bioimaging and biolabeling because of their successful cellular internalization and bright intracellular luminescence.

Fig. 5 Fluorescence images of HeLa cells alone (a); fluorescence (b) and bright-field optical images (c) of HeLa cells after incubation for 8 h with 0.1 mg mL⁻¹ of TbPO₄:Eu nanoparticles; a representative fluorescence image of HeLa cells stained with TbPO₄:Eu nanoparticles from an internal slice (d).

To reveal the cellular internalization process of nanoparticles upon time, the intracellular luminescence intensity was recorded as a function of incubation time (Fig. 6). It can be seen that the luminescence intensity increases with time during the first 6 h, and then reached a plateau at 8–10 h. This indicates that more and more nanoparticles were able to permeate into the cell membrane with time since the intracellular luminescence intensity is proportional to the number of nanoparticles taken up by cells. The uptake of nanoparticles significantly increases for the first 6 h. Subsequently, the uptake gradually slowed and reached the saturation state.

Fig. 6 Intracellular luminescence intensity as a function of incubation time.

The surface of the as-synthesized TbPO₄:Eu nanoparticles is negatively charged due to the presence of citrate groups. Generally, the anionic molecules and structures are difficult to enter the membrane with the negative charged surface because of electrostatic repulsion. The mechanism of cellular internalization of nanoparticles is not clear so far. Some researchers stated that the cellular uptake of nanoparticles involves a receptor-mediated endocytosis.^10,27,28 It has been reported that serum proteins from biological media, such as bovine serum albumin (BSA), can nonspecifically coat the surfaces of nanoparticles, leading to all nanoparticles surface bearing the same effective charge, regardless of their initial surface charge.^26,27 On this basis, it was considered that the surface adsorbed BSA facilitated the uptake of particles into human cancer cells, such as HeLa or HT-29, viareceptor-mediated endocytosis arising from cellular recognition of these proteins. To support this speculation, we observed the cellular internalization of nanoparticles at low temperature (4 °C). Endocytosis is known to be energy dependent and could be hindered at low temperature.²⁹ It is found that the fluorescence intensity of cells incubated at 4 °C decreases by about one order of magnitude as compared with that of cells treated at 37 °C (Fig. S3, ESI†), indicating a significant hindrance of cellular internalization at 4 °C. The above result confirms that the receptor-mediated endocytosis dominates over the cellular internalization of the as-synthesized TbPO₄:Eu nanoparticles, while the passive diffusion process of migration of nanoparticles seems a minor contribution.

2.4 Photostability

It should be pointed out that one of the advantages of rare-earth-doped luminescent nanoparticles as biological labels is their long fluorescence lifetime and stable photoluminescence features. The photostability of Eu³⁺-doped TbPO₄ nanoparticles was investigated by means of time-sequential scanning of the cells. After 300 s of continuous illumination with a laser, no obvious change was observed in the photoluminescence intensity (Fig. 7). In fact, no photobleaching of our present Eu³⁺-doped TbPO₄ nanoparticles was observed under further long-term illumination. These findings prove that the Eu³⁺-doped TbPO₄ nanoparticles are highly resistant to photobleaching compared to conventional luminescent labels such as organic dyes.⁶ Long luminescence lifetime and high photostability are advantageous for long-time observations in biological cases.

Fig. 7 Photoluminescence intensity as a function of illumination time.

2.5 Cytotoxicity

Only nontoxic nanomaterials are suitable for bioimaging and cell labeling. To verify whether these luminescent TbPO₄:Eu nanoparticles are biologically nontoxic and biocompatible, the particle-induced cytotoxic effects on HeLa cells were evaluated by conducting MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. The viability of untreated cells was assumed to be 100%. Fig. 8a shows the concentration effect of TbPO₄:Eu nanoparticles on cell viability after incubation for 8 h. It can be seen that, TbPO₄:Eu nanoparticles show almost no toxicity upon incubation with HeLa cells. The cell viability remains as high as 92% even after incubation with 0.1 mg mL⁻¹TbPO₄:Eu for 8 h. Fig. 8b shows the incubation time dependence on the cell viability at a given TbPO₄:Eu concentration (0.1 mg mL⁻¹). The observed cell viability shows only a slight decrease with the incubation time and remains as high as 90% even after incubation with 0.1 mg mL⁻¹TbPO₄:Eu nanoparticles for long as 72 h. Low toxicity of rare-earth-based inorganic nanoparticles further demonstrated the potential of their use as biological labels.

Fig. 8 In vitro cell viability of HeLa cells incubated with TbPO₄:Eu nanoparticles at different concentrations (a) for periods ranging from 0 to 72 h (b).

3. Experimental

3.1. Synthesis of materials

Rare earth nitrate (Tb(NO₃)₃ and Eu(NO₃)₃) stock solutions of 0.2 M were prepared by dissolving the corresponding metal oxide in concentrated HNO₃ at elevated temperature. In a typical procedure using citric acid (CA) as ligand, 20 mL of Tb(NO₃)₃ (0.2 M) solution was added to 20 mL of aqueous solution containing 8 mmol of citric acid. The solution, turning turbid due to the formation of the terbium citrate complex, was kept under vigorous stirring for 30 min. Subsequently, an aqueous solution containing 4.4 mmol of (NH₄)₂HPO₄ was added. The final pH of the mixture was adjusted to about 1–2 by adding aqueous ammonia (NH₄OH). After additional agitation for 15 min, the solution was transferred into a Teflon bottle held in a stainless steel autoclave, which was sealed and heated at 150 °C for 20 h. After cooling down the autoclave to room temperature, the precipitation was separated by centrifugation, washed with deionized water and ethanol several times, and finally dried overnight under air at 60 °C. Eu³⁺-doped terbium phosphate samples were obtained under the same conditions by replacing a fraction of Tb(NO₃)₃ by Eu(NO₃)₃.

3.2. In vitro biological experiments

3.2.1. Cell culture. HeLa cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, penicillin (100 units mL⁻¹) and streptomycin (100 mg mL⁻¹). Cells were cultured with the complete medium in 5% CO₂ at 37 °C. For all experiments, cells were harvested from subconfluent cultures by the use of trypsin and were resuspended in fresh complete medium before plating.

3.2.2. Cell uptake. In a typical procedure, 7.5 × 10⁴cells were plated in a 35 mm Petri dish for 4 h to allow the cells to attach. After the cells were washed twice with phosphate buffered saline (PBS), TbPO₄:Eu nanoparticles were added to the Petri dishes. After incubation for 4–24 h, the cells were washed several times with PBS to remove the remaining particles and dead cells, and then observed under a laser scanning confocal microscope (TCS SP5, Leica), operating at around a 380 nm excitation wavelength using a tunable Chameleon XR laser system.

3.2.3. Cell viability assay. In vitro cytotoxicity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) reduction. In a typical procedure, 2000 cells were plated in 96-well plates for 24 h to allow the cells to attach, and then incubated with TbPO₄:Eu nanoparticles in 5% CO₂ at 37 °C. At the end of the incubation time, the medium containing TbPO₄:Eu nanoparticles was removed, MTT solution (200 mL, diluted in a culture medium to a final concentration of 1 mg mL⁻¹) was added, and the mixture was incubated for another 4 h. The medium was then replaced with dimethyl sulfoxide (200 mL), and the absorbance was monitored with a microplate reader at a wavelength of 570 nm. The cytotoxicity was expressed as the percentage of cell viability compared to that of untreated control cells.

3.3. Structure and morphology characterization

The X-ray powder diffraction (XRD) data were collected on an X'Pert MPD Philips diffractometer (CuKα X-radiation at 40 kV and 50 mA). The patterns were measured in the 2θ range from 10° to 60° with a scanning step of 0.02°. Scanning electron microscopy (SEM) images were recorded using a FEG-SEM Hitachi SU-70 microscope operating at 4 kV at a working distance of 2–3 mm. For the SEM observations, samples were prepared without any carbon coating simply by depositing some powder onto a double gluing tape. High resolution transmission electron microscopy (HRTEM) investigations were carried using a JEOL 2200FS microscope. Samples for TEM investigations were prepared by first dispersing the particles in ethanol under assistance of ultrasonication and then dropping 1 drop of the suspension on a copper TEM grid coated with a holey carbon film.

3.4. Photoluminescence characterization

The luminescence in the ultraviolet/visible (UV/vis) spectral range was recorded at room temperature with a modular double grating excitation spectrofluorimeter with a TRIAX 320 emission monochromator (Fluorolog-3, Jobin Yvon-Spex) coupled to an R928 Hamamatsu photomultiplier, using the front face acquisition mode. The excitation source was a 450 W Xe arc lamp. The emission spectra were corrected for detection and optical spectral response of the spectrofluorimeter and the excitation spectra were corrected for the spectral distribution of the lamp intensity using a photodiode reference detector. Fluorescent lifetime measurements were made under a pulsed excitation at 355 nm from the third harmonic of a Nd:YAG laser. It was with a line width of 1.0 cm⁻¹, pulse duration of 10 ns, and repetition frequency of 10 Hz. The absolute emission quantum yields were measured at room temperature using a quantum yield measurement system C9920-02 from Hamamatsu with a 150 W xenon lamp coupled to a monochromator for wavelength discrimination, an integrating sphere as a sample chamber, and a multichannel analyzer for signal detection. Three measurements were made for each sample so that the average value is reported. The method is accurate to within 10%.

4. Conclusions

Luminescent TbPO₄ nanoparticles were synthesized via a citric-acid-mediated hydrothermal route. The as-synthesized nanoparticles are hexagonal prisms with a diameter of about 60–80 nm. The excitation spectra and lifetime measurements confirmed the presence of Tb³⁺-to-Eu³⁺ energy transfer in Eu³⁺-doped TbPO₄. The efficient Tb³⁺-to-Eu³⁺ energy transfer leads to four-fold increase of the absolute luminescence quantum yield, compared to undoped TbPO₄. We conducted in vitro biological experiments using human cervical carcinoma HeLa cells. The cellular uptake of TbPO₄:Eu nanoparticles based on receptor-mediated endocytosis was observed, leading to bright intracellular luminescence. The cellular uptake dependence on the incubation time was investigated, revealing that the cellular uptake significantly increases first and then reaches a plateau. The cell viability assay and the photoluminescence intensity dependence upon irradiation time reveal very low cytotoxicity and high photostability of the luminescent particles. This work demonstrates the advantages of the use of rare-earth-based nanocrystals in biological labeling agents because they combine high emission quantum yield, long luminescence lifetime, low cytotoxicity and high photostability.

Acknowledgements

This study was partly supported by the Grant-in Aid for Scientific Research of the JSPS and the World Premier International Research (WPI) Center Initiative on Materials Nanoarchitronics (MANA), MEXT, Japan, the WCU (WorldClass University) program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (R31-10013) and FCT project (PTDC/CTM/73243/2006). NS wishes to thank the JST PRESTO program for financial support.

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Footnote

† Electronic supplementary information (ESI) available: XRD pattern of the as-synthesized TbPO₄ (Fig. S1), room temperature excitation spectra of pure TbPO₄ and Eu³⁺-doped TbPO₄ (Fig. S2), and the luminescence intensity of cells incubated with TbPO₄:Eu nanoparticles at 37 °C and 4 °C, respectively (Fig. S3). See DOI: 10.1039/c0nr00673d

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