Facilely Fabricated Luminescent Nanoparticle Thermosensor for Real

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Facilely-fabricated luminescent nanoparticle thermosensor for real-time microthermography in living animals * Ferdinandus, Satoshi Arai, Shinji Takeoka, Shin'ichi Ishiwata, Madoka Suzuki, and Hirotaka Sato ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00320 • Publication Date (Web): 27 Sep 2016 Downloaded from http://pubs.acs.org on October 2, 2016

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Facilely-fabricated luminescent nanoparticle thermosensor for real-time microthermography in living animals

Ferdinandus,†,ǁ Satoshi Arai,‡,ǁ Shinji Takeoka,‡,§,⊥ Shin’ichi Ishiwata,‡,§,# Madoka Suzuki,*,‡,§ and Hirotaka Sato*,†

†

School of Mechanical & Aerospace Engineering, Nanyang Technological University, Singapore

‡

Waseda Bioscience Research Institute in Singapore (WABIOS), Singapore

§

Organization for University Research Initiatives, Waseda University, Japan

⊥

Department of Life Science & Medical Bioscience, Faculty of Science & Engineering, Waseda University, Japan

#

Department of Physics, Faculty of Science and Engineering, Waseda University, Japan

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ABSTRACT This paper presents a high-sensitivity luminescent nanoparticle thermosensor capable of realtime microthermography in living organism. Microthermography, or microscopically visualizing the temperature-distribution within living cells, tissues and organisms, is a promising technology to explore various physiological activities at the micro-scale. Using a facile nano-precipitation method, we fabricated a polymer-nanoparticle embedding EuDT, a thermosenstive highluminescence-emitter dye molecule, and rhodamine 800, a temperature less-sensitive luminescent molecule excitable with low energy light. The nanoparticle thermosensor was largely exempted from the background noise which is the undesired luminescence from the target biological sample, enabling direct acquisition of luminescence intensities from the thermosensor within the specified area of 68 µm x 68 µm on the muscle tissue of a living insect, i.e. real-time microthermography, without the need of subtracting background noise. Thus, we successfully mapped out the temperature shift due to the animal’s voluntary heat production. The nanoparticle thermosensor capable of in vivo temperature-mapping must be a useful biological thermographic technology to explore microscopic heat productions in living organisms.

KEYWORDS: thermography; in vivo thermography, luminescent thermosensor; nanoparticle thermosensor; thermogenesis; bioimaging; fluorescent thermosensor; temperature sensitive dye

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Microthermography in living animals, visualizing the temperature distribution in cells, tissues, and organisms with high spatial resolution in the order of 100 µm or smaller, allows for locating thermogenesis sites. To visualize temperature shift in such biological samples, varieties of temperature sensitive luminescent materials (probes) including organic molecular dyes,

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quantum dots,10-14 proteins,15-20 and dye-embedded polymers,21-29 whose luminescence intensity, lifetime or

spectrum-shift varies due to temperature shift, have been designed and

synthesized.30 To date, most of the probes have been tested to demonstrate 4-8, 10-11, 16-18, 21-22

luminescence thermometry in cultured cells,

but just a few were

examined in tiny living organisms, C. elegans15 and D. Melanogaster (fruitfly) larvae,23-24. Even in these living organism based examination, they have succeeded to visualize the ‘externally’ induced temperature shifts (animals were heated by laser or hot plate). To more practically use the luminescence thermometry in vivo to detect intrinsic physiological or animal’s voluntary heat productions (‘internally’ induced temperatureshifts),

undesired

luminescence

from

sample

tissues

(autofluorescence)

as

a

background noise has been a major hurdle which must be subtracted in most cases.31-37 Also, to expand the scope of the luminescence thermometry beyond the cellular scale up to the living organisms, busy procedures in the probe preparation should be simplified to be accessible for researchers even without expertise in chemical synthesis. In this study, we proposed and demonstrated a facile fabrication of a polymer nanoparticle thermosensor embedding specially selected luminescent molecules which reduced the contribution of background noise to allow real-time microthermography without the need of background subtraction. As a result, the nanoparticle thermosensor achieved high spatial and temperature resolution mapping out of temperature shift caused by the muscles of a living organism to monitor the heat production and transfer within the muscles.

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MATERIALS AND METHODS

Chemicals

Poly(styrene-co-methacrylic acid) (PS-MA) (Mw: 38000), polyvinylalcohol (PVA) (Mw:1300023000), and rhodamine 800 were purchased from Sigma-Aldrich. Eu-tris(dinaphthoylmethane)bis-trioctylphosphine oxide (EuDT) was synthesized according to the previous literature (Shinsei Chemical Company Ltd).38

Fabrication and characterization of nanoparticle thermosensor

The ratiometric nanoparticle thermosensors (RNTs) were fabricated by nano-precipitation method

21-22

. PS-MA (10 mg), EuDT (1.5 mg) and rhodamine 800 (0.01 mg) were dissolved in

tetrahydrofuran (THF, 1 mL) and were then added into a stirring aqueous solution of PVA (160 mg in 8 mL). The mixture was then left stirring at 1000 rpm for 1 hour at room temperature. Afterwards, the mixture was heated to 60 °C to evaporate the THF solvent completely. The resultant suspension was then purified using a Sephadex PD column (GE Healthcare) in order to remove the free dyes and the polymers which were not forming particles. The hydrodynamic diameter of the fabricated nanoparticle was measured by using a Zetasizer ZSP (Malvern). The luminescence properties of the particle were recorded by utilizing a fluorescence spectrophotometer (Hitachi F-2700) while monitoring the sample temperature with a thermocouple (TES-1310 type-K, TES Electrical Electronic Corp.). The Field Emission Scanning Electron Microscopy (FESEM) was performed by FESEM JOEL 7600F with an acceleration voltage of 5.0 kV and High-Angle Annular Dark-Field Scanning Transmission Electron Microscopy (HAADF-STEM) was performed by FEI Titan 80/300 S/TEM with an accelerating voltage of 200 keV. The UV-visible spectroscopy was performed by Jasco V-670 Spectrophotometer. The effect of pH on the stability of the RNT was studied by dissolving the RNT solution into the MOPS (3-(N-morpholino)propanesulfonic acid) buffer (pH = 5-10), adjusted using HCl or KOH while monitoring the pH using a pH meter (F-72, Horiba).

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Stereo-microscope setup and in vivo experiments

Microscopy experiments were conducted by using an Olympus MVX10 Macro Zoom System Microscope with objective lens MVPLAPO 1X, NA 0.25. Microscopic images were captured using an EM-CCD camera (iXon3 897; Andor Technology). FF01-405/10 and FF01-640/14 excitation filters were used to excite EuDT and rhodamine 800, respectively. FF01-515/588/700 and FF01-665/150 barrier filters were used to collect the emissions from both dyes. A Di01R405/488/543/635 dichroic mirror was also used to selectively separate the excitation and emission lights. A Lumencor Spectra X light engine was utilized as the excitation light source. The size of the observation field was 3.50 × 3.50 mm in 512 × 512 pixels. Two-dimensional images were acquired with an exposure time of 30 msec and time series images were obtained with a rate of 3 seconds per frame or 0.33 Hz. Infrared thermography (IRT) measurement was done by using a Fluke Ti400 infrared camera with an observation field size of 320 × 240 (76800) pixels and a frame rate of 9 Hz. The in vivo experiments using Dicronorrhina derbyana beetle (as in Figures 4 and 5) were conducted by tethering the beetle on the top of a stick which was fixed to a metallic base as shown in Figure S1. To prevent the movement of the beetle during observation, the front and mid legs of the beetle were dissected, leaving the two hind legs with the tarsus removed. The beetle’s dorsal longitudinal muscle (DLM) was exposed by removing the cuticles above the muscle. A buffer solution containing the RNTs was then cast to cover the whole exposed area of the muscle. For the in vivo calibration of the RNT on the muscle as in Figure 4, the muscle was heated up by a diode-pumped solid-state 980 nm CW laser connected with a flexible optical fiber to a collimating lens (5 mm ⌀ spot size) (Viasho) (Figure S1). The water in the muscle absorbs the light at 980 nm, and consequently, the muscle was externally heated up by the laser

39

. The

temperature of the muscle was raised in a step-wise manner by increasing the laser power with 2 minutes waiting interval between different laser powers, ensuring a homogeneous temperature over the muscle by IRT. Microscopic images of the muscle were acquired after the 2 minutes waiting interval. The muscle was then allowed to cool down naturally while the laser

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power was reduced in a similar step-wise manner every 2 minutes interval. The pre-flight preparation of the beetle as in Figure 5 was elicited by a mechanical stimulus, i.e., gently pinching one of the hind legs with a pair of tweezers. As a reaction of the beetle to the stimulus, the flight muscles were activated for warming up. The beetle was then left to naturally cool down to room temperature. These in vivo experiments were completed within two hours after the RNT loading. The in vivo calibration was carried out after the temperature measurement of the natural preflight preparation to avoid any influence of the heating-cooling process by laser illumination on the pre-flight preparation process which is the major physiological event of interest in this paper. In the in vivo calibration, the wide-view temperature of the muscle during the heating and cooling was carefully monitored using infrared camera in order to prevent the muscle damage due to the laser illumination. The muscle temperature during the calibration was set to cover the temperature range of the muscle during the pre-flight preparation process. The beetles were then tested to perform repeated pre-flight preparation process after the calibration process to further confirm the undetectable damage caused by the calibration process to the muscle.

Data analysis

For the in vitro spectrum analysis, the maximum emission intensity was obtained for both EuDT at 615 nm (I615) and rhodamine 800 at 710 nm (I710). The ratio of intensity of EuDT to that of rhodamine 800 (I615/I710) was then plotted against the varied temperatures (Figure 2d). For the in vivo ratiometric analysis, ImageJ software (National Institutes of Health) was used to analyze the microscopic images after capture. Signal from the muscle in both the EuDT and rhodamine 800 channels was measured by calculating the mean intensity within the different regions of interest (ROIs), 68 µm x 68 µm each. To avoid accidentally failed RNT-load in vivo, we confirmed the success of the RNT loading by checking whether the signal was higher than 7000 (a.u.) for the in vivo experiments as in Figures 4, 5, S10, and S12. The temperature sensitivity of the RNT is defined from the gradient of the plot of averaged intensity ratio (I615/I710, n = 24 ROIs) against the varied temperatures. The temperature resolution is defined as the

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standard deviation (SD) of the ratio (I615/I710) at each temperature divided by the overall temperature sensitivity of the RNT. In Figures 5 and S12, the intensity ratio (I615/I710) at every temperature point was normalized by the ratio at 22.9 °C for each ROI. For Figure S12, the shift in the normalized intensity ratio (I615/I710), ∆R, is calculated as difference of the normalized intensity ratio at 22.9 °C and at 26.7 °C so as to unify the condition between the in vivo calibration and the pre-flight preparation.

Study animal

We used Dicronorrhina derbyana (Coleoptera: Scarabaeidae) as our insect model. The size and weight of a D. derbyana beetle are approximately 40 mm and 3 g, respectively. The beetles were kept in separate plastic terrariums (20 cm × 15 cm × 15 cm) with tissue paper as the base and were fed with a cup of sugar jelly (Lai Bao Food Co., Ltd) weekly. The temperature and relative humidity in the terrariums were maintained at 23 °C and 50 %, respectively

40

. After any

experiments, the tested beetles were returned back to the terrarium, cared and fed under the same conditions as before the experiments until they ended their lives. The use of beetles is permitted by the Agri-Food & Veterinary Authority of Singapore (AVA, HS code: 01069000, Product code: ALV002). Invertebrates including insects are exempted from the ethics for animal experimentation according to National Advisory Committee for Laboratory Animal Research (NACLAR) Guidelines.

RESULTS AND DISCUSSION

In vitro performance of the nanoparticle thermosensor In this study, we facilely prepared polymer nanoparticles containing temperature sensitive and less-sensitive dyes as thermosensors to be loaded into tissues of small living animals (Figure 1). The ratiometric principle, where the ratio of the intensity of the temperature sensitive dye to that of the less-sensitive one is used as the thermo-barometer, was applied to detect in

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vivo temperature shift within the animal in order to acquire the luminescence intensity shift solely due to the temperature shift by eliminating the intensity shift due to the displacement of animal

22, 24, 30

. To have such a ratiometric nanoparticle thermosensor (RNT), a temperature

sensitive dye Eu-tris(dinaphthoylmethane)-bis(trioctylphosphine oxide) (EuDT) and a lesssensitive dye, rhodamine 800 as the reference dye were embedded in hydrophobic poly(styrene-co-methacrylic acid) (PS-MA) matrix through the facile nano-precipitation method 21, 41

(Figure 1b)

. The overall preparation process was completed within a few hours at low

temperature (room temperature or 60 °C) and under ambient pressure. EuDT has high photostability and relatively low phototoxicity due to not ultra violet ray but blue light excitation (400 nm)

25

. EuDT showed strong emission such that the autofluorescence contribution is small

(Figure 3a,d). EuDT has a relatively low quantum yield and low stability in the aqueous solution (Figure S2), but these drawbacks are improved by embedding EuDT into a hydrophobic polymer

30, 38, 42

. Rhodamine 800 is excitable at a low energy visible red light (635 nm) and

shows emission at 710 nm, apart from the strong autofluorescence emission in the shortwavelength (green to blue) range

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.

The hydrodynamic diameter of the RNT was measured as 70 ± 23 nm (Figure 2a), which agrees with the High-Angle Annular Dark-Field Scanning Transmission Electron Microscopy observation (Figure 2b). The UV-vis absorption spectrum (Figure S3) confirmed that the dyes were embedded in the polymer. The temperature sensitivity was examined by measuring the emission spectrum of the RNT suspension at varied temperatures (Figure 2c). The ratio of the luminescence intensity of EuDT to that of rhodamine 800 (I615/I710) was plotted against the varied temperatures as shown in Figure 2d. The temperature sensitivity, defined as the gradient of the I615/I710 plot, is –0.43/°C (–4.0%/°C relative to 21.5 °C, Figure S4) in the temperature range from 21.5 °C to 32.0 °C which is comparable to or higher than that of earlier reported RNTs

1, 21-25

. Emission spectrum and temperature sensitivity of the RNT in a wider temperature

range (21.5 °C to 44.0 °C) for other application is available in Figure S5. The RNT also displayed reversible luminescence ratio in response to the varied temperatures (Figure S6) and even in N2 saturated solution (Figure S7). The RNT also showed stable luminescence intensity

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under long time observation at high temperatures (Figure S8) and under different pH conditions (Figure S9). These results combined imply the leakage of the dyes from the RNT was negligible.

In vivo performance of the nanoparticle thermosensor We demonstrated the validity of the fabricated RNT for in vivo temperature monitoring in a living animal, particularly to monitor heat production in flight muscles of D. derbyana beetle (Figure 1). The use of EuDT and rhodamine 800 reduces the undesired contribution of the autofluorescence from tissues, eliminating the need of background subtraction and allowing direct use of the luminescence intensity for ratiometric analysis. We examined the RNT in vivo, i.e., we loaded the RNT onto the dorsal longitudinal muscle, a major flight muscle of the insect. We measured the luminescence intensity of the muscle in both EuDT and rhodamine 800 channels before and after the RNT loading (Figure 3). Although the muscle exhibited some autofluorescence in both channels (see ‘before’ the RNT loading in Figure 3), it accounts to approximately 10-15% of the total luminescence intensity after the RNT loading. The RNT correctly and regularly functioned even in vivo to detect temperature shifts in the muscle. The RNT was tested and calibrated in vivo, i.e., the luminescence intensities were measured as a function of temperature shift in an externally heated muscle. The RNT-loaded muscle was exposed to a 980 nm laser, which was absorbed by water molecules in the muscle and raised the temperature (Figure S1). Once the laser was shut off, the muscle naturally cooled down. The luminescence intensities in the EuDT and rhodamine 800 channels were acquired from the multiple regions of interest (ROIs, 68 µm x 68 µm each as in Figure 4a). The mean I615/I710 of 24 ROIs for the heating-cooling process is plotted against the temperature shift in Figure 4b. The temperature sensitivity, the gradient of the I615/I710 curve (red plots in Figure 4b), is –0.09/°C (–2.6%/°C relative to 21.5 °C, Figure S10a). Since the absolute temperature sensitivity is mostly influenced by given experimental setup

22, 24

, in order to fairly compare the

temperature sensitivities between different experimental setups, the absolute temperature sensitivity should be normalized by the I615/I710 at a temperature point (21.5 °C in this study). Such a normalized temperature sensitivity was low in vivo (–2.6%/°C, Figure S10a, and additional five trials using different beetles in Figure S10b-f) compared to the in vitro case

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(–4.0%/°C, Figure S4). The RNT reversibly responded to temperature shifts even in vivo (the red and blue plots in Figures 4b and S10), indicating that photobleaching of either dye was negligible. The temperature resolution (δT), defined as the standard deviation (SD) of the intensity ratio divided by the sensitivity of the RNT (–0.09/°C, Figure 4b) 44-45 was around 1.0-1.4 °C throughout the examined temperature range (the black plots in Figure 4b), which is sufficient to detect temperature shift due to the animal’s voluntary heat production in the muscle.

In vivo temperature monitoring in living muscle during animal’s voluntary heat production The RNT-loaded beetle was examined for detecting the in vivo temperature shift due to physiological heat production, particularly during the ‘pre-flight preparation’ which was elicited by a mechanical stimulation of a leg leading to the activation of the flight muscle (Figure 5a, middle). We then allowed the beetle to naturally cool down the muscle (Figure 5a, right). The shift of the normalized intensity ratio (I615/I710, normalized by the ratio at 22.9 °C), ∆R, and the temperature shift (∆T) measured by IRT are plotted against the elapsed time after the pre-flight preparation was elicited (Figure 5b). The trend seen in all the ∆R curves is same as that in the ∆T curve (temperature went up first and then down), indicating the RNT successfully tracked the internally induced temperature shift due to the animal’s voluntary heat production. Besides, there was no detectable influence of the RNT loading on the beetles’ survivorship and muscular activity as the RNT-loaded beetles were alive and exhibited the pre-flight preparation, at least two days after being loaded (Table S1, Figure S11). Note that the in vivo temperature monitoring experiments (as in Figures 4 and 5) were completed within just two hours after the RNT loading. The variance in the normalized ∆R (corresponding to the heat balance at every ROI) during the period that the temperature measured by IRT varied from 22.9 °C to 26.4 °C among the different 24 ROIs (Figure S12a) is significantly large in the pre-flight preparation compared to the in vivo calibration (Figure S12), suggesting the temperature shift is heterogeneous in preflight preparation. The RNT demonstration in this study (Figure 5) implies the heterogeneous

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heat production or heat transfer along the muscle fibre axis. Further investigation and comparison would reveal more details of heat production and heat transfer in flight muscles.

CONCLUSIONS The facilely fabricated RNT containing EuDT temperature sensitive dye and rhodamine 800 less-sensitive dye (reference) enables direct ratiometric in vivo temperature monitoring and mapping with a high temperature and spatial resolutions, without the need of background subtraction owing to the reduced autofluorescence from the tissue. The RNT exhibits high temperature sensitivity both in vitro and in vivo. The RNT successfully detected the in vivo temperature shifts at localized sites in the muscle of a living animal either due to an external heat source or due to the animal’s voluntary muscle-activation (pre-flight preparation). Owing to the high spatial resolution with fair temperature resolution, the RNT system demonstrated in this study provides a promising in vivo thermographic technology to explore the microscopic heat production and transfer in varieties of living organisms. Furthermore, the RNT system can be combined with other luminescent indicators and thereby allows for simultaneous monitoring of multiple physiological activities coupled with in vivo temperature shift.

ASSOCIATED CONTENT Supporting Information Supporting Information Available: The following files are available free of charge. Temperature rise of the beetles’ flight muscle during the pre-flight preparation after RNT loading. Microscopic setup, EuDT Spectrum in aqueous condition, UV-vis absorption spectrum of the RNT, in vitro normalized luminescence ratio of the RNT, in vitro normalized luminescence ratio of the RNT at 21.5 °C to 44.0 °C, Reversibility of the RNT response against heating-cooling cycle, Reversibility of the RNT under N2 saturated solution, Stability of the RNT at higher temperatures, Stability of the RNT in MOPS buffer with different pH, Infrared thermogram of the

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beetle 2 days after RNT loading, in vivo normalized luminescence ratio of the RNT, Spreading of the normalized ∆R values in the in vivo experiments (PDF).

AUTHOR INFORMATION Corresponding Authors * M. Suzuki. E-mail: [email protected] * H. Sato. E-mail: [email protected] Author Contributions ǁ

These authors contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work is supported by Nanyang Assistant Professorship (NAP, No. M4080740, to H.S.), A*STAR-JST (Agency for Science, Technology and Research, Singapore and The Japan Science and Technology Agency, Japan) joint grant (No. M4070198, to S.I. and H.S.), Japan Society for the Promotion of Science (JSPS) KAKENHI grant (No. 15K05251 and 26107717, to M.S.), and The Murata Science Foundation (to S.A.). The authors thank Mr. Vo Doan Tat Thang of School of MAE, NTU for the fruitful discussion on the pre-flight preparation mode of the beetle and Mr. Poon Kee Chun of School of MAE, NTU and Dr. Lin Ming of IMRE, A*STAR for the help on electron microscopy experiments.

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FIGURE CAPTIONS

Figure 1. (a) Ratiometric nanoparticle thermosensor (RNT) was loaded into the dorsal longitudinal muscle, a flight muscle (black dashed square, inset) of a beetle Dicronorrhina derbyana. (b) Schematic representation of the nanoparticle. The temperature sensitive dye, EuDT (red circles) and the reference dye, rhodamine 800 (blue squares) were both embedded in the PS-MA matrix, coated with the hydrophilic layer of PVA (light blue shell).

Figure 2. In vitro performance of the RNT. (a) Size distribution of the RNT, average size = 70 ± 23 nm (DLS measurement). (b) High-Angle Annular Dark-Field Scanning Transmission Electron Microscopy (HAADF-STEM) image of the prepared RNT. (c) EuDT emission spectra (λex = 400 nm) of the RNT at varied temperatures (from 21.5 °C to 32.0 °C). Inset: Rhodamine 800 emission spectra (λex = 635 nm) of the RNT. (d) The ratio of luminescence intensity of EuDT to that of rhodamine 800 (I615/I710) was plotted (n = 3 trials, different colours represent independent trials) against the varied temperatures. The temperature sensitivity or the gradient of the averaged data (black line) is –0.43/°C (y = –0.43x + 19.56, R² = 0.99).

Figure 3. Pseudocolor images displaying the luminescence intensity of flight muscle before (left) and after (right) loading of RNT: (a) EuDT, (b) Rhodamine 800 channels, and (c) Ratiometric (EuDT/Rhodamine 800) image derived from (a) and (b). Each pseudocolor image is accompanied by the color scale corresponding to the measured luminescence intensity or intensity ratio as indicated. The yellow rectangles indicate the region of interest (ROI) for intensity analysis shown in the histograms in (d). (d) The histograms showing the luminescence intensity measured from the ROI depicted for before (blue bars) and after (red bars) the RNT loading.

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Figure 4. In vivo calibration of the RNT loaded onto the flight muscle of living D. derbyana beetle, which was externally heated up and naturally cooled down. (a) Beetle flight muscle shown with 24 ROIs for the ratiometric intensity analysis as in (b). The size of every ROI is 68 µm x 68 µm. (b) The mean luminescence intensity ratio (I615/I710) from the ROIs is plotted against varied temperatures. The ratio shifts during the heating (20.7 to 28.4 °C, red circles) and cooling (28.4 to 21.4 °C, blue circles) were plotted with the SD (vertical bars, n = 24 ROIs). The temperature sensitivity of the RNT, or the gradient of the curve during the heating, is –0.09/°C (y = –0.09x + 5.4, R2 = 0.99). The temperature resolution (δT) defined as SD divided by the temperature sensitivity (0.09/°C), was plotted at every temperature as black circles. The in vivo calibrations were carried out after the temperature measurement of the natural pre-flight preparation process.

Figure 5. In vivo temperature monitoring and mapping of RNT loaded flight muscle of a living D. derbyana beetle. The muscle was activated and the pre-flight preparation was triggered which leads to the warm up of the muscle as shown in (a) IRT thermography. The tiny black rectangles show the position and scale of the area of (b). (b) Temperature shifts detected by RNT loaded into the flight muscle at the different ROIs. The normalized ratio (R, I615/I710, relative to 22.9 °C) was plotted against the elapsed time from the triggering of the pre-flight preparation in the right panel. The size of every ROI is 68 µm x 68 µm. The temperature shifts measured by IRT (∆T, black line, from 22.5 °C to 26.7 °C and return back to 22.5°C) are plotted at the lowest trace.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Facilely Fabricated Luminescent Nanoparticle Thermosensor for Real

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