Dual-Emissive Phosphorescent Polymer Probe for Accurate

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Biological and Medical Applications of Materials and Interfaces

Dual-Emissive Phosphorescent Polymer Probe for Accurate Temperature Sensing in Living Cells and Zebrafish Using Ratiometric and Phosphorescence Lifetime Imaging Microscopy Huajie Zhang, Jiayang Jiang, Pengli Gao, Tianshe Yang, Kenneth Yin Zhang, Zejing Chen, Shujuan Liu, Wei Huang, and Qiang Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01565 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 7, 2018

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Dual-Emissive Phosphorescent Polymer Probe for Accurate Temperature Sensing in Living Cells and Zebrafish Using Ratiometric and Phosphorescence Lifetime Imaging Microscopy Huajie Zhang,†, || Jiayang Jiang,†, || Pengli Gao,† Tianshe Yang,† Kenneth Yin Zhang,† Zejing Chen,† Shujuan Liu,†,* Wei Huang,†,‡,* and Qiang Zhao,†,* †

Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key Laboratory

for Biosensors, Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, 9 Wenyuan Road, Nanjing 210023, China. E-mail: [email protected], [email protected]. ‡

Shanxi Institute of Flexible Electronics (SIFE), Northwestern Polytechnical University (NPU),

Xi'an 710072, China. E-mail: [email protected]. KEYWORDS: iridium(III) complexes, lifetime imaging, phosphorescent polymer probe, ratiometric imaging, temperature sensing

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ABSTRACT

Temperature plays an important part in many biochemical processes. Accurate diagnosis and proper treatment usually depend on precise measurement of temperature. In this work, a dualemissive phosphorescent polymer temperature probe, composed of iridium(III) complexes as temperature sensitive unit with phosphorescence lifetime of ~500 ns and europium(III) complexes as reference unit with lifetime of ~400 µs, has been rationally designed and synthesized. Upon the increase of the temperature, the luminescence intensity from the iridium(III) complexes enhances, while that from the europium(III) complexes keep unchanged, which makes it possible for the ratiometric detection of temperature. Furthermore, the polymer also displays a significant change in emission lifetime accompanied with the temperature variation. By utilizing the laser scanning confocal microscope (LSCM) and time-resolved luminescence imaging (TRLI) systems, ratiometric and time-resolved luminescence imaging in Hela cells and zebrafish have been carried out. Notably, the intensity ratio and long-lifetime based imaging can offer higher sensitivity, decrease the detection limit and minimize the background interference from biosamples.

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1. Introduction The variation of the intracellular temperature is associated with many important cellular activities, including the mass transfer, energy conversion, signal transduction, and the metabolism, division and apoptosis of cells. Therefore, monitoring intracellular temperature provides the possibility of understanding the underlying cause of diseases, and offers the diagnostic and therapeutic methods for clinical applications.1-4 Contact thermometer (thermoelectric couple) and non-contact thermometer (infrared thermometer) as physical measurement of temperature usually have low spatial resolution and sometimes may cause exogenous lesion. On the contrary, luminescent probes which exhibit significant luminescence alteration with temperature variation can provide noninvasive approach for intracellular temperature determination.5-8, Up to now, many luminescent materials including organic dyes,9,10 polymers,11-16

quantum

dots,17-19

nanoclusters,20,21

upconversion

nanoparticles22-24

and

fluorescent proteins25 have been reported for visual and pinpoint detection of temperature in vitro and in vivo. However, intensity-based temperature sensing may be disturbed by many factors such as the excitation and emission efficiency, as well as the inhomogeneous distribution of the probes. In contrast, self-calibrated ratiometric probes, which reflect the variation of the microenvironment conditions by the change of the luminescence intensity ratio at two wavelengths, lead to the improvement of accuracy and the decrease of the detection limits.26-34 Phosphorescent transition metal complexes (PTMCs) have achieved raising attention in biological area during the last few years. These complexes are labled as variable emission wavelength, high quantum efficiency, long emission lifetime, large Stokes shift and good photostability.35-43 Utilizing phosphorescence lifetime imaging microscopy (PLIM), the longlived phosphorescence of PTMCs provide a possibility to exclude the interference from short-

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lived spontaneous fluorescence and tissue scattering during bioimaging, enabling the improvement of the imaging contrast, detecting sensitivity, spatial and time resolution.44-49 In this work, we aimed to develop a long-lived phosphorescent probe P-Ir-Eu for monitoring temperature in vitro and in vivo. An acrylamide-based thermosensitive polymer probe has been designed and synthesized (Figure 1). In the polymer, N-isopropyl acrylamide (NIPAM) was employed as the thermosensitive unit, (3-acrylamidopropyl) trimethylammonium (APTMA) was introduced to improve water solubility and cellular uptake, and phosphorescent Ir(III) complex served as the responsive luminophore. When the temperature is below the lower critical solution temperature (LCST), the hydrogen-bond interaction between amide groups and H2O in PNIPAM endows the polymer with good solubility in aqueous solution. Upon increasing the temperature, the hydrogen-bond interaction is weakened owning to the molecular thermal motion, which results in the reduction of water solubility.50 Thus, upon increasing temperature from 20 to 42 °C, the hydrophilic polymer backbone transforms to hydrophobic type, resulting in a decrease in microenvironment polarity. Then, the change of polarity leads to the variation in Ir(III) complex centered phosphorescence intensity and emission lifetime (Figure S1). Especially, the long emission lifetime of Ir(III) complex endows the polymer the ability for photoluminescence lifetime imaging in living cells and zebrafish. Moreover, as a luminescence reference and temperature insensitive unit, the f-f transition forbidened Eu(III) ion has been introduced to assist in monitoring temperature. The occurrence of energy transfer between the metal-to-ligand charge transfer (3MLCT) excited state and 5D0 state effectively realizes the sensitization process from Ir(III) complex to Eu(III) center and easily extends the excitation of Eu(III) complex to visible region.51-54 In addition, the millisecond lifetime of Eu(III) complex is also beneficial for TRLI in cells and zebrafish. Therefore, the dual-emissive phosphorescent polymer probe (P-Ir-Eu)

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containing Ir(III) and Eu(III) complex has been demonstrated for sensitive and reversible temperature sensing via self-calibrating ratiometric imaging and lifetime imaging in Hela cells and zebrafish.

2. Results and Discussion 2.1. Design, Synthesis and Characterizations As displayed in Scheme 1, the synthesis routes of polymer probe have been provided. The synthesis procedures of monomer Ir(dfpp)2(apa) (Ir) and monomer Eu(tta)3paa (Eu) were described in Supporting Information. The target polymer P-Ir-Eu was acquired through highly efficient free radical polymerization. The polymer was reprecipitated for three times in diethyl ether and was furtherly purified by dialysis for 1 day. The APTMA units were incorporated to increase the biocompatibility and the population of cellular uptake. Moreover, owing to the hydrophilia of polymer chain, the poor water-soluble metal complexes acquire good water dispersibility, which reduce the possibility of phosphorescence self-quenching. Moreover, the control polymers containing Ir(III) or Eu(III) complexes only (P-Ir and P-Eu) were also designed and synthesized for comparition. The fourier transform infrared spectra of the polymer showed successful polymerization of monomers (Figure S2). The band at 1645 cm-1 of the polymer is assigned to −CO− stretching. By contrast, the −CO− signal of NIPAM, APTMA, Ir and Eu locates at 1658, 1661, 1658 and 1660 cm-1, respectively, indicating that all the monomers have been polymerized into the backbone through free radical polymerization reaction. The average molecular weight (Mn) of P-Ir, P-Eu and P-Ir-Eu are 7900, 10400 and 11100, respectively. Via the gel permeation chromatography (GPC) test and using polystyrene standards as calibration curve, the polydispersity indexes (PDI) of the polymers in tetrahydrofuran (THF)

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solution were measured as 1.95, 2.00 and 2.05 respectively (Table S1). The real contents of different components were also provided in Table S1. The mole ratio of the NIPAM and APTMA units in polymer was calculated from 1H NMR spectrum of the NIPAM-ATPMA copolymer. The contents of the Ir(III) and Eu(III) complexes in polymer were determined from the absorption spectra of their monomers (Figure S3). The variation between feeding ratio and actual content is due to the steric effect and radical reactivity of the monomers. Then, the LCST was evaluated to be 30 °C via measuring the transmittance of P-Ir-Eu at different temperatures (Figure S4). In addition, the spherical particle morphology of P-Ir-Eu in PBS was observed by transmission electron microscopy (TEM). As shown in Figure 2a, the smallest particles were about 40 nm and the sizes of the larger ones range from 50 to 120 nm, which resulted from the temperature change induced aggregation during sample preparation (30 °C). The hydrodynamic diameter measured by dynamic light scattering (DLS) confirmed that the average diameter was approximately 10 nm at 20 °C and reached 107 nm at 30 °C (Figure 2b). The diameter of the polymer changed slightly after 2 weeks (Figure 2d), which demonstrated good stability of the polymer. As indicated in Figure 2c, the average diameter suddenly increased when the temperature arrived at 30 °C, which meant the poly(N-isopropylacrylamide) units reached the LCST. This data demonstrates that P-Ir-Eu shows small diameter and good dispersibility at low temperature, because of the hydrogen bonding between amido linkages in polymer and water molecules. As the temperature rises around the LCST, hydrogen bonding is impaired and the water molecules surrounding the polymer become free. Hydrophobic interaction among P-Ir-Eu leads to chain assemblage and the reducing of the microenvironment polarity around iridium(III) complex. 2.2. Photophysical Properties

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The absorption and photoluminescence (PL) spectra of P-Ir, P-Eu and P-Ir-Eu were shown in Figure 3a. From the absorption spectrum of P-Ir, the intense peak below 350 nm is originated from 1π–π* transitions of the dfppy and hpa ligands. The relatively weak absorption bands at 350–400 nm is attributed to the absorptions of singlet metal-to-ligand charge transfer (1MLCT) and ligand-to-ligand charge transfer (1LLCT) from Ir(III) complex. Owning to the intense spinorbit coupling effect contributed from heavy atoms, the triplet metal-to-ligand charge transfer (3MLCT) and ligand-to-ligand charge transfer (3LLCT) could be observed over 400 nm. P-Eu displayed a broad absorption band from 240 to 400 nm that is mainly assigned to the 1π–π* transitions of the tta ligand. Under excitation at 405 nm, P-Ir-Eu exhibited a broad emission across 450–650 nm and several characteristic emission peaks at 541, 581, 592, 615, and 653 nm, which are ascribed to the emission of Ir(III) complex and Eu(III) complex, respectively. The triplet energy level of Ir unit was approximately 21300 cm-1, which was evaluated from lowtemperature emission spectrum of P-Ir-Eu (Figure S5) and matched well with the 5D0 energy level of Eu ion (17500 cm-1).54 The highly efficient sensitization process has been realized in the polymer and the luminescent quantum efficiency of the polymer has been measured to be 0.63 at room temperature. As expected, P-Ir and P-Eu exhibited the characteristic emission of Ir and Eu according to the emission spectra. With the increase of excitation wavelength from 405 to 488 nm, the characteristic emission of Eu(III) ion in P-Ir-Eu was capable to be excited (Figure 3b). On the contrary, P-Eu could not be excited directly absent from the iridium sensitizer (Figure 3c). These results implied that Ir unit is capable for sensitizing Eu unit in the polymer and efficiently extending its excitation wavelength to visible area. 2.3 Temperature Detection of P-Ir-Eu in Aqueous Solution

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The response of P-Ir-Eu, P-Ir and P-Eu to temperature in phosphate buffer solution (PBS) was studied. For P-Ir-Eu, the polymer chains gradually aggregated as the temperature increasing from 20 °C to 42 °C and became hydrophobic, causing the decrease of polarity in microenvironment and the enhancement of the radiative transition population from Ir units in polymer (Figure 4a). The emission intensity of Eu units, however, was insensitive to temperature. The result was consistent with the response of P-Ir and P-Eu to temperature (Figure S6). The ratio of the phosphorescence of Ir units at 470 nm to that of Eu units at 615 nm in P-Ir-Eu was determined to be 0.13 at 20 °C, which increased to be 1.18 at 42 °C. What’s more, the intensity ratio linearly increased by 9 folds (Figure 4b) with the temperature resolution from 0.18 °C to 0.52 °C, which indicated the sensitivity and accuracy of the thermometer. The change of ratio value was reversible when the solution underwent 20 cycles between 20 °C and 42 °C (Figure 4c). The reversibility of P-Ir-Eu was illustrated under the illumination of the 405 nm laser (Figure S7). A slight fluctuation of luminescence intensity at the same temperature was detected due to the unstable source power. However, the luminescence intensity ratio of the two channels well eliminated the error of the source power. Considering the complexity of in vivo environment, the influence of concentration, ionic strength, pH value, and biomolecules on the polymer was investigated. The results (Figure S8) indicated that the intensity ratio (470 nm/615 nm) was almost independent of ionic strength (aqueous KCl, CaCl2, MgCl2), pH value (6-8) and polymer concentration (0.01 w/v% - 0.1 w/v%). In order to verify the anti-interference ability of P-Ir-Eu to biomolecules, the test in different concentration of BSA solution was carried out and the ratio was hardly influenced. Therefore, the polymeric thermometer has been demonstrated to exhibit the high selectivity, reversibility and sensitivity for sensing temperature. Besides, naked eye recognition to temperature change was realized. The luminescence color of P-Ir-Eu in PBS

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changed obviously from red to white and further to blue-green, which was shown in the chromaticity coordinates (Figure S9). The relationship between luminescence lifetime and temperature was investigated when monitored at 470 and 615 nm. The luminescence decay curve was plotted in Figure 4d. From 20 °C to 42 °C, the phosphorescence lifetime at 470 nm varied from 240 ns to 480 ns and that at 615 nm maintained at about 550 µs. The evident change in lifetime results in the high differentiation from short-lived autofluorescence through PLIM techniques. 2.4 Temperature Imaging of P-Ir-Eu in Living Cells Before P-Ir-Eu was applied in bioimaging and biosensing, the photostability and cytotoxicity in cells are the vital factors to be concerned.55 The photostability of the polymer in cells was measured by laser scanning confocal microscope and the result was shown in Figure S10. The strong ability against photobleaching is beneficial for long-time temperature monitoring in vitro and in vivo. Then, cell toxicity test has been investigated with Hela cells. The cancer cells were incubated in 6-well plates with different polymer concentration at 37 °C in a incubator of 5% CO2 for 24 h. Annexin V-FITC/PI kit was applied in this experiment and the cells were dyed according to the product’s protocol and washed with PBS for three times before using FlowSight imaging flow cytometer for the analysis. The statistical values for live, apoptosis, and death cell from flow cytometer analysis were given in Figure S11. The cell viability still achieved almost 80% even incubated with the maximum concentration (0.8 mg/L) of the polymer. These results imply the good biocompatibility and the low cytotoxicity of the polymer. The intracellular temperature sensing utilizing P-Ir-Eu was carried out by LCSM system. A heating stage and a digital thermocouple were used to control the temperature of extracellular buffer, and the luminescence signals were collected through two channels (green channel of 460-

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540 nm and red channel of 580-630 nm). To realize the endocytosis of P-Ir-Eu to cells, Hela cells were incubated with 0.3 mg/mL polymer at 37 °C for 1 h. Upon heating from 25 °C to 35 °C, the phosphorescence intensity in 460-540 nm increased (Figure 5a), and that in 580-630 nm almost unchanged, which is in keeping with the results obtained in solution. As shown in Figure 5b, the phosphorescence intensity ratio of 470 nm to 615 nm varied from 1.1 to 1.4. The rising tendency of the ratio values was also similar to that in PBS. Thus, the polymer showed excellent sensitivity and accuracy for thermal sensing in living cells. Moreover, different incubation concentration (0.06, 0.3, 0.9, 1.2 mg/mL) of P-Ir-Eu in Hela cells was tested (Figure S12). These results demonstrated that the luminescence ratio is independent of the incubation concentration, which leads to accurate intracellular temperature determination by ratiometric readout. To further demonstrate the anti-interference ability in complex environment, the photoluminescence lifetime imaging of P-Ir-Eu in Hela cells has been carried out. The photoluminescence lifetimes of the green channel (480 ± 40 nm) at 25 °C, 30 °C, and 35 °C were 286 ns, 344 ns, and 390 ns, respectively (Figure 5c), and that of red channel (609 ± 54 nm) kept approximately 135 µs (Figure 5d). The lifetime variation tendency is in accordance with that in aqueous solution. The lifetime variation tendency is in accordance with that in aqueous solution. The disparity of the luminescence ratio between that in water and in vivo was attributed to the different detecting instruments and complicated intracellular environment. These data demonstrated that high signal-to-noise ratio and sensitive temperature imaging can be realized with the polymer via luminescence ratiometric imaging and phosphorescence lifetime imaging techniques. 2.5 Temperature Imaging of P-Ir-Eu in Zebrafish

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Next, the sensing capability of P-Ir-Eu in zebrafish larva was investigated. Owing to 87 percent identical at the genetic level with human, zebrafish model is widely used in studying cancers, diabetes, genetic cardiovascular diseases.56,57 Herein, we have demonstrated that P-IrEu is an excellent polymetric thermometer in vivo. Aqueous P-Ir-Eu was injected into the heart of zebrafish (Figure 6a). After 2 h incubation, the luminescent signal of P-Ir-Eu could be detected around the heart part of zebrafish. Within 12 h, the polymer has been distributed in the whole body due to its excellent biocompatibility (Figure S13). With the increase of environmental temperature from 25 °C to 30 °C, the photoluminescence intensity around 480 nm enhanced, and that around 615 nm remained almost unchanged (Figure 6b). Due to the stability and biocompatibility of P-Ir-Eu in vivo, the variation tendency of intensity coincided with that measured in PBS solution. Due to the strong autofluorescence in zebrafish, however, the distinction between luminescence signal of P-Ir-Eu and autofluorescence in zebrafish was not evident. Because the lifetime of autofluorescence in zebrafish was significantly shorter than that of the polymer, we can confirm the position of P-Ir-Eu in zebrafish via PLIM technique (Figure 6c), which endowed the images with higher signal-to-noise ratio. As expected, the tendency of decay lifetime variation of the polymer in zebrafish is also similar with that in PBS solution and living cells. The average lifetime of the green channel (480 ± 40 nm) at 20, 25 and 30 °C increased to be 223, 267, and 310 ns respectively, and that of red channel (609 ± 54 nm) kept around 130 µs (Figure 6d). The photoluminescence lifetime imaging has provided a higher temperature detection resolution than photoluminescence imaging.

3. Conlusion

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In summary, a dual-emissive phosphorescent polymer probe containing both phosphorescent Ir(III) complex and Eu(III) complex has been developed for temperature sensing. The probe exhibits good water solubility, biocompatibility and excellent reversibility to temperature. Compared with the LCST (24 °C) in our previous work,14 the value (30 °C) of P-Ir-Eu in this work is higher, which is more suitable for temperature detection in living system. The temperature resolution of P-Ir-Eu has been improved to be 0.18-0.52 °C, so that it is beneficial for more accurate temperature detection. Moreover, Eu(III) complex is introduced into the polymer thermometer, which exhibits longer emission lifetime than the previous polymer thermometer. The elongated emission lifetime makes this polymer thermometer more favorable for lifetime-based temperature sensing and imaging. The ratiometric imaging and PLIM of temperature variation in Hela cells and zebrafish have been realized successfully, exhibiting high accuracy and effective elimination of interference from the background fluorescence. It is worth mentioning that many kinds of long-lived units can be chosen as the ratiometric pairs by incorporation into the polymer backbone. We believe that this class of long-lived temperature probe could be used for studying the energy equilibrium in normal or pathological cells, via monitoring temperature variation caused by the environment or physiological activities. It also provides the possibility to investigate many diseases at cellular level, permitting the development of novel diagnostic and therapeutic methods.

4. Experimental Section 4.1. Experimental Methods Synthesis of monomers and other intermediate products, experimental device information, cell culture and imaging methods, etc. have been provided in Supporting Information.

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4.2. Synthetic Section The synthetic routes of P-Ir and P-Eu were the same as that of P-Ir-Eu. The Preparation of Dual-emissive Phosphorescent Polymer (P-Ir-Eu). A mixture of N-isopropyl acrylamide (NIPAM, 347.3 mg, 3.07 mmol), 3-acrylamido-N, N, N-trimethylpropan-1-aminium chloride (APTMA, 32.3 mg, 0.16 mmol), Ir(dfpp)2(apa) (20.0 mg, 26.6 mmol), Eu(tta)3(paa) (14.2 mg, 13.3 mmol) and azodiisobutyronitrile (AIBN, 10.7 mg, 65.2 mmol) were dissolved in tetrahydrofuran (THF, 2.0 mL) and dimethylformamide (DMF, 0.2 mL). The reaction was stirred at 80 oC and kept for 12 h under nitrogen atmosphere. The mixture was added dropwise to 150 mL diethyl ether to afford P-Ir-Eu after cooling down the reactor. The precipitated polymer was gathered by filtration and was furtherly purified by 1 day dialysis. Yield: 35.4%. GPC (THF, polystyrene standard): Mn = 11100, Mw/Mn = 2.05.

ASSOCIATED CONTENT Instrumental information, measuring approaches and cell experiments; Synthesis procedure, MALDI-TOF, 1H NMR,

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C NMR and FTIR results of Ir, Eu monomers and polymer P-Ir, P-

Eu, P-Ir-Eu; PL spectra of P-Ir and P-Eu; CIE chromaticity diagram of P-Ir-Eu; The reversibility test and anti-interference ability of P-Ir-Eu in vitro; Cytotoxicity experiments with P-Ir-Eu. The Supporting Information is free on the website (http://pubs.acs.org).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected]

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*E-mail: [email protected] Author Contributions ||

H. J. Zhang and J. Y. Jiang contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We acknowledge financial support from the National Program for Support of TopNotch Young Professionals, National Natural Science Foundation of China (21671108 and 51473078), and Priority Academic Program Development of Jiangsu Higher Education Institutions (YX03001).

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Figure 1. (a) Chemical structures and design strategy of P-Ir-Eu. (b) Thermosensitive mechanism of P-Ir-Eu.

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Scheme 1. The synthetic routes of polymer P-Ir, P-Eu and P-Ir-Eu.

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Figure 2. (a) The TEM image of P-Ir-Eu. (b) The DLS data of P-Ir-Eu at 20 °C and 30 °C. (c) The hydrodynamic diameter change of P-Ir-Eu at different temperatures (20 °C-42 °C) in PBS. (d) The average diameters of P-Ir-Eu in PBS solution at 25 °C within 2 weeks.

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Figure 3. (a) The normalized absorption and emission spectra of P-Ir, P-Eu and P-Ir-Eu. (b) PL spectra of P-Ir-Eu with the excitation wavelength changing from 405 nm to 488 nm. (c) Excitation spectra of P-Eu and P-Ir-Eu monitored at 615 nm. All the spectra were acquired at 25 °C.

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Figure 4. (a) Photoluminescence response of P-Ir-Eu to temperature from 20 to 42 °C in PBS; (b) Plots of temperature-dependent ratio of emission intensity at 470 nm to that at 615 nm and temperature resolution (left axis and right axis respectively). (c) Reversibility of the temperaturedependent luminescence intensity of P-Ir-Eu (0.1 w/v%) in PBS for 20 times between 20 oC and 42 oC. (d) Relationship between lifetime and temperature of P-Ir-Eu in PBS solution with bandpass filter (480 ± 40 nm) and (609 ± 54 nm), respectively. The excitation wavelength was 405 nm.

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Figure 5. (a) CLSM images of Hela cells treated with P-Ir-Eu at 25 °C, 30 °C and 35 °C. (b) Luminescence intensity collected from different channels in zebrafish at 24 °C, 29 °C and 34 °C. The green and red channels were acquired by collecting the luminescence 460-540 nm, 580-630 nm, respectively. (c) The PLIM images and (d) the lifetime distribution curve of P-Ir-Eu in Hela cells collection at 480 ± 40 nm (above) and 609 ± 54 nm bandpass filter (below) at 25 °C, 30 °C and 35 °C. The excitation wavelength was 405 nm.

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Figure 6. (a) CLSM images of zebrafish treated with P-Ir-Eu at 20 °C, 25 °C and 30 °C. (b) Luminescence intensity collected from different channels in zebrafish at 24 °C, 29 °C and 34 °C. The green and red channels were acquired by collecting the luminescence 460-540 nm, 580-630 nm, respectively. (c) The PLIM images and (d) the lifetime distribution curve of P-Ir-Eu in zebrafish with 480 ± 40 nm bandpass filter (above) and 609 ± 54 nm bandpass filter (below) at 25 °C, 30 °C and 35 °C. The excitation wavelength was 405 nm.

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