Ratiometric Nanothermometer Based on Rhodamine Dye

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Ratiometric nanothermometer based on Rhodamine dye-incorporated F127melamine-formaldehyde polymer nanoparticle: preparation, characterization, wide-range temperature sensing, and precise intracellular thermometry Youshen Wu, Jiajun Liu, Jingwen Ma, Yongchun Liu, Ya Wang, and Daocheng Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03366 • Publication Date (Web): 20 May 2016 Downloaded from http://pubs.acs.org on May 23, 2016

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ACS Applied Materials & Interfaces

Ratiometric nanothermometer based on Rhodamine dye-incorporated F127-melamine-formaldehyde polymer nanoparticle: preparation, characterization, wide-range temperature sensing, and precise intracellular thermometry Youshen Wu‡, Jiajun Liu‡, Jingwen Ma, Yongchun Liu, Ya Wang, and Daocheng Wu* Key Laboratory of Biomedical Information Engineering of Education Ministry, School of Life Science and Technology Xi'an Jiaotong University Xi'an, 710049, P. R. China. KEYWORDS: intracellular thermometry; ratiometric thermometer, Rhodamine dyes, wide range temperature sensing, melamine-formaldehyde resin

ABSTRACT: A series of fluorescent nanothermometers (FTs) was prepared with Rhodamine dye-incorporated Pluronic® F-127–melamine–formaldehyde composite polymer nanoparticles (R–F127–MF NPs). The highly soluble Rhodamine dye molecules were bound with Pluronic® F127 micelles and subsequently incorporated in the cross-linked MF resin NPs during hightemperature cross-link treatment. The morphology and chemical structure of R–F127–MF NPs

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were characterized with dynamic light scattering, electron microscopy, and Fourier-transform infrared (FTIR) spectra. Fluorescence properties and thermo-responsivities were analyzed using fluorescence spectra. R–F127–MF NPs are found to be monodispersed, presenting a size range of 88–105 nm, bright fluorescence, and high stability in severe treatments such as autoclave sterilization and lyophilization. By simultaneously incorporating Rhodamine B and Rhodamine 110 (as reference) dyes at a doping ratio of 1:400 in the NPs, ratiometric FTs with high sensibility of 7.6 %·°C−1 and a wide temperature sensing range of −20 °C to 110 °C were obtained. The FTs exhibit good stability in solutions with varied pH, ionic strengths and viscosities, and have similar working curves in both intracellular and extracellular environments. Cellular temperature variations in Hela cells during microwave exposure were successfully monitored using the FTs, indicating their considerable potential applications in the biomedical field.

1. Introduction As a common and important physical quantity, temperature performs a key function in miscellaneous scientific fields, and measurement of temperature at the microscale and nanoscale is of significance in the fields of industry1, nanotechnology2, and biomedical sciences3. The luminescence properties of certain materials are related to temperature. By analyzing the emission of materials, surrounding temperature could be obtained, thus providing a fast, noninvasive approach for temperature sensing4-7. Recently, numerous FTs have been developed for nanoscale thermometry applications6, 8. Various fluorescent nanoparticle (NP) materials, such as nanodiamond3, 9, semiconductor quantum dots (QDs)10-12, fluorescent gold nanoclusters13, lanthanide NPs14-15, and thermo-


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responsive nanogels16-18, have been used for FTs, and some of these materials have been successfully applied for specific nanoscale thermometry applications, including intracellular temperature sensing19-21. Particularly, fluorescent temperature sensors with wide-sensing ranges are needed for nanoscale thermometry applications6. FTs with higher resolution (sensitivity) and better thermo-stability are of significant importance in the research of microfluidic system, biotechnology and aerodynamic. For example, using Rhodamine B dye as molecular temperature sensor, thermal imaging of Joule-heating caused by electrokinetically pumping buffers in microfluidic channels were obtained, it was found that the temperature variation ranges in the Tshaped microfluidic system could larger than 40°C22. The DNA amplify process of Polymerase chain reaction (PCR) is widely used in molecular biology, during the thermal cyclings of PCR, the solution temperature should be accurately controlled in the wide range of room temperature to 94-96°C. However, the temperature sensing ranges of reported FTs remain limited by the thermostability of the used material. For example, the temperature sensing range of green fluorescent protein (GFP) is 20 °C to 50 °C

23

; the sensing ranges of nanogel FTs are

approximately ±15 °C around the transition temperature of the gel

16-18, 24-25

; Albers et al.

developed ratiometric FT with QDs, which has sensing range of 20°C to 40 °C

12

; Li et al.

developed FT with silicon NPs with a sensing range of 0 °C to 60 °C26. Although some of the FTs prepared with inorganic fluorescent materials, such as lanthanide NPs, can act in wider temperature range (50 K to 300 K), ultraviolet (UV) excitation is required. UV may lead to strong background in biological samples14-15,

27

, thereby limiting the application of these

materials’ biomedical fields because common confocal microscope and flow cytometer are usually equipped with excitation laser of longer wavelength (405, 488, and 533 nm)

28

. In

particular, the sensing properties of certain FTs may change in a complex sensing environment


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(in solution, cells, and tissues) and deteriorate during severe treatments, such as autoclave sterilization at 121 °C and storage8, 13, 16-17, 29. Thus, FTs with stable accuracy, wide sensing range, good biocompatibility, and proper excitation and emission are urgently needed. Recent studies above are focused on the fluorescent nanoparticle (NP) materials and their fabrications. In fact, several organic fluorophores, such as Rhodamine dyes, exhibit thermo-responsive properties, which can be utilized in thermometry applications 22, 30-31. Rhodamine dyes exhibit high molar-extinction coefficients and quantum yields, bright fluorescence emissions, and good stability, which have been used for the fabrication of miscellaneous fluorescence sensors32-35. These dyes have also been used for the preparation of functional fluorescent materials36-38. Most Rhodamine dyes show temperature-insensitive emissions, whereas the fluorescence intensity of Rhodamine B largely decreases with the increase in temperature31, 39, this unique emission characteristic makes it an effective molecular thermometer dye, which has been applied for wide-range (20 °C to 90 °C) temperature mapping of the fluid22, 40. Moreover, Rhodamine B is used together with a reference fluorophore to acquire ratiometrically changed emissions40. By conjugating Rhodamine B to a fluorescent conjugated polymer, Ye et al. prepared ratiometric FTs, which have been successfully used for intracellular temperature sensing of Hela cells

41

. However, covalent bonding between Rhodamine B and

polymer leads to a significant decrease in temperature sensing sensitivity; thus, the intensity ratio is only 2.2 times the variation within the temperature range of 10 °C to 60 °C41. In addition, owing to the high solubility of Rhodamine B in water (15 g·L−1), the stability of Rhodamine Bincorporated fluorescent NPs prepared by precipitation or soaking cannot easily meet the requirements of thermometry applications, and the dye would leak from fluorescent particles during storage and use37,

42

. Thus, for the preparation of bright ratiometric FTs, suitable


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preparation methods to simultaneously incorporate Rhodamine B and reference dyes effectively to work in a wide temperature range in biomedical fields remain lacking.

Scheme 1. Schematic illustration of the preparation of the R-F127-MF-NP FTs and their intracellular thermometry. Melamine–formaldehyde (MF) resin, a transparent (for UV–visible light) polymer that has highly cross-linked interior structure and good thermostablity, is considered as an excellent matrix for fluorescent material preparations43-44. In previous studies, we developed an organic sol–gel process to prepare monodispersed MF particles45 and investigated the soluble dye incorporation and particle formation mechanism of fluorescent encoded MF microspheres43. In this process, soluble MF pre-polymer was first prepared by the polymerization of melamine and paraformaldehyde in solution, and branch-structured pre-polymer molecules were mixed and conjugated with soluble dyes easily. Through high-temperature crosslinking treatment, MF pre-


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polymer molecules were further polymerized into microspheres; as a result, soluble dyes, such as Rhodamine B, can be quantitatively encapsulated in MF microspheres. However, this method can only fabricate micro-sized fluorescent particles as reported. Although MF NPs can be prepared with the addition of the surfactant, added surfactants such as sodium dodecyl sulfate also hinder the binding interaction between the dyes and the pre-polymer, thus hampering the preparation of fluorescent MF NPs. However, no suitable method to prepare Rhodamine B MF NPs has yet been reported. Notably, block copolymer Pluronic® F-127 can be used as the surfactant for the preparation of resorcinol–formaldehyde (RF, an analog resin of MF) NPs

46

.

Pluronic®F-127 and Rhodamine dyes can also form uniform a dye–micelle inclusion in water and can be further coated with silica in a sol–gel process

47-48

. In consideration of the sol–gel

particle formation mechanism of MF NPs, this dye–micelle inclusion may also be incorporated in the formed MF NPs. Inspired by this finding, we developed a three-step method to prepare Rhodamine dye-incorporated Pluronic®F127–MF composite NPs (R–F127–MF NPs), as Scheme 1 shows. Rhodamine dyes are initially incorporated with F127 micelles. The obtained dye–micelle inclusion was further polymerized with melamine and paraformaldehyde to prepare the dye–micelle–MF polymer complex. Under acid catalysis, the complex became cross-linked at high temperature of 100 °C, leading to the formation of monodispersed fluorescent NPs. Notably, Pluronic®F127 can not only help the formation of MF NPs in this three-step method but also bind with Rhodamine dyes to form dye–micelle inclusion and then form the dye– micelle–MF polymer complex. As a result, Rhodamine dyes could be firmly bound in prepared NPs. By incorporating the sensor dye (Rhodamine B) and reference dye (Rhodamine 110) simultaneously in the NPs, ratiometric FTs prepared temperature related changed emission signature, which can act in the wide temperature range of −20 °C to 150 °C without aggregation


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or dye leakage. These FTs present good stability toward various environmental factors, including pH, ionic strength, and viscosity, and can endure severe treatments, such as autoclave sterilization and cryopreservation. More importantly, the prepared FTs also present higher sensitivity and good biocompatibility, which can be applied for intracellular thermometry. The FTs also exhibit similar working curves in both intracellular and extracellular environments, indicating the possibility of precise absolute temperature measurement of cells associated with diseases. Cellular temperature changes during microwave exposure at varied power have been successfully measured. This microwave heating process was monitored in real time using a confocal microscope system, thus providing the feasibility of biological applications in a wide temperature range. 2. Materials and Methods 2.1 Materials Pluronic® F-127 (F127), Rhodamine B (RB), Rhodamine 110 (R110), dimethyl sulfoxide (DMSO), and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, USA). Melamine, paraformaldehyde, glycerol, and hydrochloric acid solution (1.0 mol·L−1) were purchased from Aladdin Chemistry Co., Ltd (Shanghai, China). All reagents were of analytical grade and used without further purification. Deionized water was used in all experiments. 2.2 Preparation of R–F127–MF NPs Rhodamine dyes incorporated F127-MF NPs (R-F127-MF NPs) with doping concentration of 1.0 µmol·g−1 were prepared as follows (using RB dye as example). One gram of Pluronic® F-127 and 2.0 mg of RB dye were mixed with 100 mL water and stirred for 2 h for the formation of the dye–micelle inclusion. A 1.3 g portion of melamine and 1.9 g of paraformaldehyde were then


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added, and the mixture was heated at 50 °C with magnetic stirring for 70 min for the formation of dye–micelle–polymer complex. The obtained dye–micelle–polymer complex solution was then filtered using three layers of filter paper to remove the insoluble impurities. This solution was then mixed with 1–3 mol·L−1 hydrochloric acid solution in a volume ratio of 1:5 and heated at 100 °C (in a boiling water bath) for 40 min to allow the formation and cross-linking of NPs. The prepared RB incorporated F127–MF NPs (RB–F127–MF NPs) were separated and washed with water using the CENTRICON® (Darmstadt, Germany) Centrifugal Filter (30,000 NMWL) for five times. The obtained NPs were then lyophilized and kept in 4 °C. In a similar process, RB NPs and R110 dye-incorporated F127–MF NPs (R110–F127–MF NPs) with varied doping concentrations were prepared. To prepare NPs containing both reference and sensing dyes (R110RB–F127–MF NPs NPs), the R110 and RB dyes are first prepared as dye–micelle inclusion with F127, and the two obtained kinds of dye–micelle inclusion solutions were then mixed in a certain ratio to prepare the dye–micelle–polymer complex. Through high-temperature crosslinking, the prepared dual dye-incorporated dye–micelle–polymer complex was then processed into NPs. The ratio of these two emissions can be adjusted by changing the mixing ratio of the two dye–micelle inclusion solutions. 2.3 Characterization Transmission electron microscopy (TEM) images of the prepared NPs were captured using a JEOL (Tokyo, Japan) JEM-2100F field-emission electron microscope with a 200 kV electron source. The hydrodynamic diameters of the samples were measured using a Malvern (Worcestershire, UK) Zetasizer Nano ZS90 DLS (dynamic light scattering) system. The Fouriertransform infrared (FTIR) spectra of the samples were measured using a Bruker Optics (Ettlingen, Germany) Tensor 27 spectrometer. The temperature-related fluorescence spectra of


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samples were measured using a HORIBA Jobin Yvon (Pairs, French) FluoroMax-4 fluorescence spectrophotometer with a Wavelength Electronics (Bozeman, USA) LFI-3751 temperature controller. The lyophilized NP samples were diluted to a mass concentration of 50 µg·mL−1 with phosphate buffer solution (PBS, pH = 7.2). For spectrum measurements in a wide temperature range of −20 °C to 150 °C, NPs were dispersed in 90 wt.% glycerol solution. Fluorescence lifetimes were measured with a 44MXs-B (LeCroy, USA) time-correlated single photon counting apparatus. The samples were excited using a Fianium SC400-4-PP (Southampton, UK) fiber laser, and results were recorded by using a PicoHarp300 (Berlin, Germany) single photon counting apparatus. Data were analyzed by using multiple exponential models. 2.4 Stability, accuracy and sensitivity analysis For verifying the stability of the prepared NPs at extreme temperatures and pressures, the fluorescent NP solution was treated using autoclave sterilization (121 °C, 103.4 kPa) and lyophilization at −80 °C. The colloidal stabilities of NPs were evaluated by their size distributions before and after treatments. The treated NPs were separated by using ultramembrane centrifugation, and the fluorescence of NPs and the supernatant were measured and compared to evaluate possible dye leakage during treatments. The influences of environmental factors on the accuracy of FTs were analyzed through the working curves (emission intensity ratio versus temperature curve) of FTs in various solutions. PBS at pH = 5, pH = 7, and pH = 9 were prepared by mixing the solutions of dipotassium phosphate and monopotassium phosphate. Solutions with different ionic strengths were prepared by 0, 150, 500, and 1000 mM of NaCl additions. Solutions of different viscosities were obtained by 20, 40, 60, and 80 wt.% glycerol addition.


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Photostabilities of the prepared FTs were analyzed by photobleaching tests. Samples of the prepared FTs and solutions of the used dyes were irradiated with a 300W high pressure mercury lamp for 4 hours, and emission intensity of the samples were measured every 30 minutes at 25°C. 2.5 Cell culture and cell viability assay H9c2 rat cardiomyoblasts from ATCC and Hela cell lines were cultured in high-glucose DMEM, which contains 10% FBS, 100 µg·m L−1 of streptomycin, and 100 U·mL−1 of penicillin, at 37 °C with 5% CO2 in a Thermo (Waltham, UK) humidified incubator. MTT assay was performed to assess the in vitro cytotoxicity of the prepared R–F127–MF NPs. H9c2 cells were first cultured for 24 h before sequential incubation with F127–MF NPs for 24 h at concentrations of 50, 100, and 200 µg·m L−1. After incubation, cells were washed with PBS and incubated with 0.5 mg·mL−1 MTT in DMEM for 1 h at 37 °C. The medium was discarded, and the generated formazan was dissolved in DMSO. The absorbance of DMSO solutions were then recorded using a Thermo Scientific (New York, USA) Multiskan GO Microplate spectrophotometer at 550 nm. 2.6 Cellular temperature monitoring during microwave exposure Hela cells were cultured in 10 cm-diameter plates with poly-D-Lysine-coated coverslips (CITOGLAS Co., Ltd., Nanjing, China) and incubated with R–F127–MF NPs for 4 h. The FT labeled cell samples were observed using Nikon (Tokyo, Japan) Nikon ECLIPSE Ti-S and ECLIPSE 80i fluorescence microscopes. The obtained cell culture coverslips were then soaked in PBS solution and placed in lidded microcuvettes for fluorescence measurements. The calibration curve was obtained by averaging the data on temperature-related emission intensity ratios of five samples. A Shengpu (Xuzhou, China) SPW-1A microwave therapy apparatus was used to generate microwave radiation. A planar spiral antenna was used for directionally heating the cell samples.


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Cell samples were prepared as culture coverslips, which were soaked in PBS solution and placed in lidded microcuvettes for simultaneous heating and fluorescence measurements. During heating, cell samples were exposed to 2450 MHz frequency microwave radiation with 10, 20, 30, and 40 W power for 6 min. For observing temperature-related ratiometric fluorescence changes of NPs in Hela cells during microwave heating process, Hela cells were cultured in 35 mm glass-bottomed Petri dishes and incubated with NPs. The obtained cell samples were heated with 40 W, 2450 MHz microwave radiation and observed using a Carl Zeiss (Oberkochen, Germany) LSM 700 laser scanning confocal microscope. Temperature changes in the culture media were monitored using a thermocouple thermometer. Laser scanning confocal microscopy images were captured with 20 mW argon-ion laser excitation at 488 nm. 3. Results and Discussion 3.1 Preparation of R–F127–MF NPs Rhodamine dyes are fluorescent dyes that exhibit bright emissions. These dyes have been used in various fluorescent functional materials. Previously studies have reported the preparation of fluorescent NPs by incorporating Rhodamine dyes in the matrix of silica and polymer materials37, 42-43. RB exhibits unique temperature-related emission intensity and has been utilized as a molecular thermometer22, 31. However, Rhodamine dyes are polar molecular ions that usually exhibit high solubility in water and other solvents. In particular, owing to high solubility in water and other solvents, RB is more prone to diffusion and leaking from the matrix material in comparison with other dyes. For example, R6G has been successfully used in the preparation of fluorescent encoded cross-linked polystyrene microspheres. Meanwhile, leakage of RB has been proven in the same matrix37. On the other hand, the temperature-related fluorescence properties


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of RB may be influenced by the covalent linkage of the dye and the matrix. The sensitivity of RB as a fluorescent temperature sensor considerably decreased, whereas the dyes were grafted to the fluorescent conjugated polymer 41. Therefore, for the preparation of RB incorporated NPs as FT, a method is required to solve the problems of leakage and decrease in sensitivity. On the basis of our previously developed organic sol–gel preparations of MF particles, we developed a novel three-step process in this study to prepare monodispersed fluorescent NPs with RB as FT. As Scheme 1 showed, RB was first mixed with block polymer Pluronic® F-127 to prepare dyemicelle inclusion; the prepared dye–micelle inclusion was further processed into dye–micelle– polymer complex by the polymerization of melamine and formaldehyde, and R–F127–MF NPs were prepared via high-temperature crosslinking treatment. In this process, RB dye molecules were evenly incorporated in the prepared F127–MF composite NPs and firmly entrapped by the highly cross-linked three-dimensional network of the MF resin, with the fluorescence properties remaining unchanged. As block polymer F127 molecules self-assemble into uniform micelles in water, the amphipathic micellar structure of F127 has been utilized for the preparation of various functional materials

46-49

. Compared with ionic surfactant, F127 is of a considerably lower critical micelle

concentration, and the obtained micelles exhibit good stability in solutions of varied pH and temperature

46, 49

. Ma et al. reported their preparation of a silica fluorescent material with F127.

Through the affinity between the dye molecules and the amphiphilic micellar structures, dyemicelle inclusion was prepared with Cy5.5 and F127, and the inclusions were subsequently encapsulated in the silica matrix by sol–gel process. In this study, the dye–micelle–polymer complex of Rhodamine dyes, F127, and MF resin polymer was prepared in a similar manner. By mixing and stirring Rhodamine dyes and F127 in water for 4 h at room temperature (25 °C), the


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dye–micelle inclusion solution was obtained. With the prepared dye–micelle inclusion solution, the dye–micelle–polymer complex was further subjected to polymerization of melamine and formaldehyde at 50 °C for 50 min. During this process, melamine and formaldehyde gradually dissolved and polymerized into soluble MF polymers, and the turbid suspension became a transparent solution. With acid catalysis, polycondensation of the hydroxymethyl terminal groups of the soluble MF polymer at high temperature resulted in crosslinking of the highly branched MF polymer into a three-dimensional network45. The prepared dye–micelle–polymer complex solution was mixed with dilute hydrochloric acid to adjust the pH to 4 and heated at 100 °C with boiling bath for 2 h. During the high-temperature crosslinking process, the transparent solution became slightly opalescent. Through DLS analysis, we found that the obtained solution contained the mass of NPs with average hydrodynamic radius of approximately 100 nm. The prepared NPs were then separated from the solution by ultrafiltration. The separated NPs were found to exhibit bright red fluorescence under 365 nm ultraviolet illumination, while the clear supernatant virtually exhibited no fluorescence. This finding indicates that the dye–micelle–polymer complex has been converted into R–F127–MF composite NPs, with most of the dyes incorporated. These results indicate the facility of the developed three-step fluorescent NP preparation process. With a Rhodamine dye doping concentration of 10 µmol·g−1, a high incorporation ratio of 99.5% was obtained, thereby proving the effectiveness of the high-temperature crosslinking process. The high-temperature treatment not only led to the formation of the highly cross-linked NP matrix but also ensured the thermostability of the produced NPs. We consider the versatility of this process, which may also be used for incorporating other fluorescent dyes with high solubility in water.


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Figure 1. A1 to A4. The fluorescence differences of the heated RB-F127-MF NPs solution sample (right) and the control (left) as it cooled from 80°C to 25°C, under 365nm UV illumination. B. TEM images of the NPs, scale bars=200 nm. C. Hydrodynamic diameter distributions of RB-NPs of different dye doping concentrations. D. FTIR spectra of the R-F127MF NPs and related materials. 3.2 Characterization of R–F127–MF NPs 3.2.1 Morphology and size distribution Solution samples of the prepared RB–F127–MF NPs were transparent and exhibited bright fluorescence under UV illumination (Figure 1A). The morphology of the prepared fluorescent NPs was analyzed by TEM. As shown in Figure 1B, the obtained NPs are highly monodispersed spherical particles with an average diameter of 90 nm. The influence of dye addition to the size of NPs was investigated by DLS analysis of samples containing different amounts of RB (Figure 1C). The RB–F127–MF NP samples that were prepared with varied additions of RB dyes were found to possess similar monodispersed size distributions, and the average diameter slightly


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increased from 88 nm to 105 nm as the doping concentration of RB increased from 0.1 µmol·g−1 to 10 µmol·g−1. Other R–F127–MF NPs possessed similar characteristics as described above. Compared with the reported dye-incorporated nanoparticle FTs prepared with poly(methyl methacrylate) 19, N-Isopropylacrylamide 16, 25, and silica 50 materials, R–F127–MF NPs prepared through this three-step process are of higher monodispersity, so that the polydispersity index (PDI) of samples prepared with different added dyes are all smaller than 0.005. In addition, the monodispersity of the prepared R–F127–MF is one order higher than that in our previously reported MF NPs prepared with addition of sodium dodecyl sulfate; the PDI of the MF NP samples is in the range of 0.08 to 0.0545. 3.2.2 FTIR spectroscopy The composition of the prepared R–F127–MF NPs was analyzed by FTIR spectroscopy analysis. In comparison with those of the MF resin and F127 powder samples, the FITR spectrum of the prepared NP sample showed certain characteristics of the blend of the two polymers. Several specific absorption bands of the MF resin appeared in the FTIR spectra of the prepared NP samples, originating from the vibration of the 1,3,5-s-triazine ring (at 1556 and 812 cm−1) and imino (-NH-; at 3408 cm−1). The absorption bands of methylene (-CH3-; at 1483 and 1157 cm−1) in the MF resin have shifted to 1462 and 1105 cm−1, demonstrating the influence of F127 in the R–F127 NPs 51. These results show that the R–F127–MF NP sample contained both MF and F127, as was in agreement with the conception of the preparation process. For samples with addition of various amounts and types of Rhodamine dyes, minimal differences in the FTIR spectra were observed, and the characteristic absorption peaks of Rhodamine dyes cannot be distinguished in the obtained FTIR spectra. This finding is attributed to significantly lower mass fraction of Rhodamine dyes in the obtained material relative to the


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matrix materials, and the IR absorptions have been covered by the matrix, especially while characteristic absorption peaks are in the vicinity of the absorptions of F127–MF matrix materials 52. 3.3 Temperature related fluorescence of RB-F127-MF NPs 3.3.1 Fluorescence of the prepared RB-F127-MF NPs As described above, the prepared RB–F127–MF NPs exhibit bright red-orange fluorescence under a UV illumination, proving the successful incorporation of the RB dye into the matrix of the NPs. However, as reported by previously studies, dye molecules may become aggregated in the silica matrix of the NP material during the dye incorporating process, and this change in aggregation state may also lead to changes in the excitation and emission properties of fluorescent dyes 53. Three-dimensional fluorescence spectroscopy provides comprehensive fluorescence properties of the material, which has been used for in-matrix state analysis of fluorescent dyes, such as R6G 36. Thus, to investigate fluorescence properties of the incorporated RB dye molecules, we measured and analyzed the prepared fluorescent NP material by 3D fluorescence spectroscopy. With excitation wavelength range of 400 to 500 and emission wavelength range of 510 to 650, three-dimensional fluorescence spectra of the NP solution and RB solution were measured and compared. As shown in Figure S1, both obtained spectra shared similar characteristics, and this similarity in both excitation and emission properties indicates the unchanged fluorescence properties of the incorporated RB in NPs.


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Figure 2. A. Emissions of RB-F127-MF NPs of varied dye doping concentrations. B. Dye doping concentration related emission peaks. C. Temperature related emissions of RB-F127-MF NPs with dye doping concentration of 1.0 µmol·g-1. D. Fluorescence versus temperature curves of the RB-F127-MF NPs of varied dye doping concentrations. Emission intensities of the RB–F127–MF NP samples prepared with different dye addition are proportional to the dye doping concentration (Figure 2A). We also found that the emission peaks also slightly red-shifted with increasing dye addition (Figure 2B). This finding can be explained by the homo-resonance energy transfer (homo RET) effect of the incorporated dye molecules 43. The fluorescence lifetime decay curves of NP samples of different RB addition were measured with a time-correlated single photon counting system. As shown in Figure S2, the average fluorescence lifetimes of the NP samples decreased with increasing incorporated dye concentration, thereby proving the occurrence of the homo-RET process. 3.3.2 Dye doping concentration-related fluorescence–temperature responsivity


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As shown in Figure 1A, the fluorescence intensity of the RB–F127–MF NP solution significantly decreased at high temperature (80 °C) and gradually recovered after cooling to room temperature (25 °C). This finding proved the temperature dependence of the fluorescence of the RB–F127–MF NPs. By contrast, as reported by Ye et al., the fluorescence–temperature responsivity of RB may largely decrease as the dye molecules were conjugated to the NP polymer matrix 41. To investigate the fluorescence–temperature responsivity of the prepared RB– F127–MF NPs, we measured the fluorescence emissions of the NP solution samples in the temperature range of 20 °C to 90 °C (at 10 °C intervals), and the obtained normalized intensity versus temperature curves were compared with that of the RB solution. As shown in Figure 2D, RB–F127–MF NPs prepared at the incorporated dye concentration of 0.1 µmol·g−1 exhibit similar fluorescence–temperature responsivity to that of the RB solution, with the normalized fluorescent intensity decreasing by a factor of 5.5 as temperature increases from 20 °C to 90 °C. Moreover, the fluorescence–temperature responsivity of the prepared fluorescent NPs was found to be related to the incorporated dye concentration. The fluorescent NPs prepared with incorporated dye concentration of 1.0 and 10 µmol·g−1 exhibited larger intensity variations of 6.2 and 10.8 times in the same temperature range. This dye incorporating concentration-related fluorescence–temperature responsivity may also be related to the homo-RET effect of the incorporated dye molecules. At high incorporating concentration of 10 µmol·g−1, the average intermolecular distance of dye molecules may be in the scale of several nanometers, the emission state of the dye molecule is highly coupled to all of its neighboring dye molecules, and any thermo-induced fluorescence quenching may thus affect more than one dye molecule. 3.4 Ratiometric temperature sensing properties of R110–RB–F127–MF NP FTs


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3.4.1 Incorporating of the reference dye The prepared RB–F127–MF NPs exhibit temperature-related fluorescence properties for NP samples prepared with higher incorporated dye concentrations. The fluorescence–temperature responsivities are even slightly higher than that of RB dye in solution, and the fluorescent NPs can be used as effective FTs. However, given that local fluorescence intensity is also related to NP concentration, the intensity of effective excitation and other inhomogeneities, absolute temperature values cannot be provided by the emission intensity of these FTs, especially in complex environments such as tissues and cells 54. By contrast, for FTs exhibiting ratiometrically changed fluorescence emissions, absolute temperature value could be obtained from the ratio of the reference and sensing fluorescence, and higher sensing accuracy was achieved 11, 14, 18-19. To prepare FTs with ratiometrically changed emission signature, we selected another Rhodamine dye, R110, as reference dye. As shown in Figure S3, R110 possesses a similar molecular structure as RB and exhibits maximum emission at the wavelength of 520 nm. The emission intensity of R110 only slightly decreased in the temperature range of 20 °C to 90 °C, and R110 has been utilized as reference dye with RB in temperature sensing. Through a similar threestep preparation process, R110 was proven to be able to form dye–micelle–polymer complexes with F127 and MF and be prepared into NPs by high-temperature crosslinking treatment. The prepared R110–RB–F127–MF NP FTs present similar size distribution and colloidal stability as NPs prepared with RB. Under excitation at 488 nm, the prepared R110–RB–F127–MF NP FTs exhibit two emission peaks at 520 and 582 nm, proving the successful incorporation of both dyes.


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Figure 3. A. Temperature related emissions of R110-RB-F127-MF NP FTs with dye doping ratio of 1:40 and total doping concentration of 1.0 µmol·g-1. B. Temperature related emissions of R110RB-NPs with dye doping ratio of 1:400 and total doping concentration of 10 µmol·g-1. C. Emission intensity ratio versus temperature curves of R110-RB-NPs of varied dye doping ratios. D. Emission intensity ratios variation of R110-RB-NPs (1:400, 10 µmol·g-1) during repeated heating and cooling. 3.4.2 Ratiometric sensing properties of the R110–RB–F127–MF NP FTs The temperature sensing properties of the prepared ratiometric FTs were further investigated by fluorescence emissions at different temperature. As shown in Figure 3A, the emission signatures of the prepared dual dye-incorporated NPs ratiometrically changed with temperature, and the emission intensity at the wavelength of 580 nm (RB) markedly decreased with the increased temperature, whereas emission intensity at 520 nm (R110) only slightly decreased at higher temperature. As a result, the ratios of the two emission peaks (Em 520/580) also increased


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with the temperature increase. Notably, as the emission intensity of the reference dye of R110 also slightly decreased at higher temperature, the obtained variation amplitude of intensity ratio is 3.4, which is smaller than the absolute intensity variation of RB (5.3 times) in the same temperature range of 20 °C to 90 °C. The amplitude of temperature-related emission intensity ratio variation also depends on the ratio of the two incorporated dyes. Compared with FTs prepared with the incorporated dye ratio of 1:40 (R110:RB) and total dye doping concentration of 1.0 µmol·g−1, FTs prepared with the dye ratio of 1:400 and total dye doping concentration of 10 µmol·g−1 showed larger amplitude of emission intensity ratio variation of 5.3 in the temperature range of 20 °C to 50 °C (Figure 3B). As larger ratio variation amplitude provides higher temperature sensing sensitivity, an incorporated dye ratio of 1:400 was selected for further experiments. 3.5 Stability, accuracy, and sensitivity of the R110–RB–F127–MF NP FTs 3.5.1 Stability of the R110–RB–F127–MF NP FTs during use and storage The thermostability and temperature sensing ranges of the most reported FTs are limited in the range of 20 °C to 60 °C, especially for FTs prepared with polymer materials 16, 19, 23, 25. To evaluate the thermostability of the prepared FTs, the FT solution was repeatedly heated and cooled in the temperature range of 20 °C to 90 °C for 10 times. During this process, the emission intensity ratios of the sample were measured as well. As shown in Figure 3D, the emission intensity ratios of the sample varied with temperature and showed high stability at the same temperature, indicating the excellent thermostability of the FTs.


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To meet the needs of biomedical applications, fluorescent NPs materials used for FT preparation should possess good colloidal stability in various treatments. Autoclave sterilization (121°C, 103.4 kPa) and lyophilization (−80 °C, 1.3–13 Pa) treatments, which involve using of extreme temperature and pressure, are frequently used in biomedical research. The colloidal and fluorescence stability of FTs in these treatments were analyzed. The results showed that the size distribution of FTs is unchanged after these treatments, indicating excellent colloidal stability. Moreover, dye leakage was not found. NP powder obtained by lyophilization showed good storage stability and can be stored for six months at −20 °C, without dispersibility and fluorescent property change.

Figure 4. Ratiometric temperature sensing properties of R110-RB-F127-MF NP FTs in solutions of varied pH (A), NaCl concentrations (B) and glycerol concentrations (C). D. Ratiometric temperature sensing property of R110-RB- F127-MF NPs in wide temperature range of -20°C to 110°C.


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3.5.2 Influence of various environment factors on R110–RB–F127–MF NP FTs As previously mentioned, micro/nanoscale thermometry environments are highly complex, and variation in several environmental factors, such as pH, ionic strength, and viscosity, may affect the accuracy of FTs

19

. With ratiometric FTs prepared with proper incorporated dye ratio, the

influences of various environment factors on the temperature sensing properties were investigated. As shown in Figure 4A, the prepared FTs showed unchanged intensity ratio versus temperature curves (working curves) in PBS at pH = 5, 7, and 9. The working curves of FTs in solutions containing 0, 150, 500, and 1000 mM NaCl also showed minimal changes (Figure 4B). The FTs also exhibited unchanged ratiometric sensing properties in 20%, 40%, 60%, and 80% glycerol solutions (Figure 4C), indicating good sensing reliability in high-viscosity environments. These results show that the prepared FTs exhibit high sensing stability in various environments, as can be explained by the isolation effect of the highly cross-linked MF encapsulation matrix on the incorporated dyes. In order to investigate the photostability of the prepared ratiometric FTs, the fluorescence emission intensity of the two incorporated dyes were measured during continuous irradiation. As showed in Figure S4A, the dye incorporated NPs and the dye solutions showed similar photobleaching trend during irradiation, their emission intensities maintained constant in the first hour, and get decreased in the subsequent time. The emission intensity of the two-dye incorporated NPs and the dye solutions all decreased to about 80% after irradiated with a 300W of high pressure mercury lamp for 4 hours. These results indicated that photobleaching effect gives certain influence to the emission intensity of the prepared FTs for continues excitation more than 1 hour. However, as showed in Figure S4B, as the used two Rhodamine dyes have similar photobleaching behaviors, their emission intensity ratio, or the ratiometric property of the FT, can hold steady for more than 3 hours. Thus, these


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results showed our R–F127–MF NPs have the better resistance of photobleaching that confirmed the photostabilities of the prepared ratiometric FTs. 3.5.3 Wide range temperature sensing property The temperature sensing range of prepared FTs was further investigated. To prevent the influence of solution boiling or freezing on fluorescence measurements, 90 wt.% glycerol solution was used for FT dispersion. As shown in Figure 4D, the prepared ratiometric FTs can work in a wide temperature range of −20 °C to 110 °C, with emission intensity ratio variation of 8.7 times and average sensitivity of 6.7. Owing to the limitation in the working range of the temperature controller, fluorescence emission of the sample at temperature exceeding 110 °C cannot be obtained. However, by heating the glycerol solution of the prepared FTs to 120, 130, 140, and 150 °C and cooling to room temperature, fluorescence emissions of the sample were found to exhibit minimal changes after the high-temperature treatments. This result indicates a high possible temperature sensing upper limit of 150 °C. 3.5.4 Sensitivity of the R110–RB–F127–MF NP FTs R110–RB–F127–MF NP FTs with high thermo-responsivity exhibit higher sensing resolutions and can be used for revealing the precise temperature distribution and evolution in nanoscale thermometry applications. Sensing sensitivities of the prepared series of RB–F127–MF NPs and R110–RB–F127–MF NPs the prepared series of RB–NPs and R110–RB–NPs were calculated and analyzed by signal variation amplitudes and average sensitivity in the related temperature ranges. As shown in Table 1, the prepared R–F127-MF FTs present a relative sensitivity of 4.9%·°C−1 to 15.4%·°C−1 in a large temperature sensing range of 20 °C to 90 °C. In addition, ratiometric R110– RB–NPs FTs with a doping ratio of 1:40 and doping concentration of 1.0 µmol·g−1 also


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Table 1. Sensitivity of the series of FTs Signal variation (multiply)

Sensitivity (%·C°-1 )

Rhodamine B solution (0.1µmol·g-1)

5.3（absolute intensity）

7.6 (20°C-90C°)

RB-F127-MF NPs (0.1 µmol·g-1)


7.8 (20°C-90C°)

RB-F127-MF NPs (1.0 µmol·g-1)


8.8 (20°C-90C°)

RB-F127-MF NPs (10 µmol·g-1)

10.（absolute intensity）

15.4 (20°C-90C°)

R110-RB-F127-MF NPs

3.4（ratiometric）

4.9 (20°C-90C°)


7.6 (20°C-90C°)


6.7 (-20°C-110C°)

(1:40, 1.0 µmol·g-1) R110-RB-F127-MF NPs (1:40, 1.0 µmol·g-1) R110-RB-F127-MF NPs (1:40, 1.0 µmol·g-1) in -20°C to 110°C

present a high average sensitivity of 7.6%·°C−1. This value is considerably higher than those of previously reported ratiometric FTs that are prepared with RB conjugated semiconducting polymer dots, which only possess a ratio variation of approximately 2.2 times and 2.6%·°C−1 sensitivity in the temperature range of 10 °C to 70 °C. In addition, sensitivities of the R–F127– MF FTs are also significantly higher than those reported ratiometric FTs prepared with QDs12, lanthanide NPs14, or GFP23, as the sensitivity of these FTs are usually in the range of 1% to 3% 45, 8

. The sensing sensitivities of R–F127–MF FTs are still lower than those of FTs prepared with

thermo-responsive nanogels, as the later FTs may possess sensitivity of 9% to 19%. However,


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the sensing range of these FTs is usually limited to ±15 °C around the phase transition temperature. 3.6. Biocompatibility and intracellular thermometry 3.6.1 MTT assay and biocompatibility of R–F127–MF NPs Compared with FTs prepared with lanthanide NPs and QDs, which usually exhibit cytotoxicity induced by free metal ions55-56, R–F127–MF FTs are polymer NPs and show advantages, such as biocompatibility. The cytotoxicity of the prepared R–F127–MF FTs was examined by MTT assay. The MTT assay was conducted on H9c2 cells with R–F127–MF NP addition of 50, 100, and 150 µg·mL−1. As shown in Figure5 A and B, significant difference in cell viability was not observed in the prepared fluorescent NPs, even at a high addition concentration of 150 µg·mL−1. After incubation, cells were found to be successfully labeled with FTs through the cellular uptake of NPs. In addition, the R–F127–MF FTs labeled cells were successfully cultured for 72 h, and cell proliferation was found to be unaffected by NP uptake. These results indicate that the prepared R–F127–MF FTs are of high biocompatibility and suitability for various biomedical applications.


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Figure 5. A. MTT assays of R-F127-MF-NPs of varied dye doping concentrations. B. MTT assays of the R110-RB-F127-MF NPs of varied doping ratios. C. Intracellular working curve of the ratiometric FT. D. Cellular temperature changes during the microwave exposure of varied power. 3.6.2 Intracellular temperature sensing property of R–F127–MF FTs Hyperthermia has recently attracted considerable attention in cancer therapy because of advantages of effectiveness and lower side effects6-8, 57. Precise measurement of temperature changes in cancer cells during hyperthermia is one of the key techniques in this therapy process 58

. As mentioned above, the prepared R–F127–MF FTs showed high sensing stability and high

biocompatibility, and these characteristics may be suitable for cellular temperature sensing in the hyperthermic processes. Intracellular temperature sensing property of the R–F127–MF FTs was further investigated with Hela cell samples. As shown in Figure S5, after incubation with slidecultured Hela cells for 4 h, FTs were all labeled within the cells. Both green (reference) and red (sensing) fluorescence could all be observed by fluorescence microscopy. Through analysis of


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the fluorescence emissions of FT labeled cell samples, we found that FTs retained an unchanged emission signature in the cells. By measuring emissions of the cell samples at different temperature, the in-cell working curve is obtained (Figure 5C). Compared with FTs prepared with fluorescent gold nanoclusters13, thermo-responsive nanogels17, 21, and molecular beacons29, which usually show altered sensing properties in cellular environments, R–F127–MF FTs exhibited unchanged working curve that is extremely close to that of FTs in PBS solution. This finding proved the high sensing accuracy of R–F127–MF FTs in intracellular thermometry and provided the possibility of precise absolute temperature values in the nanoscale environment. Thus, slight absolute temperature changes, which are closely associated with diseases of inflammation59, cancer60-61 cellular thermogenesis, and heat transfer studies19, could be precisely measured. 3.6.3 Monitoring of cellular temperature during microwave exposure Microwave irradiation is one of the most commonly used heating sources for hyperthermia. With designed antennas, microwave irradiation can be used for directional heating of the tumor with programmable power and time62. Moreover, we found that cell death induced by microwave irradiation exhibit certain unique features; thus, immunohistochemical antigenicity of threatened cells is well-preserved in this process63. With the obtained in-cell working curve of the R–F127– MF NP FTs, microwave irradiation-induced temperature changes of the FT labeled cell samples were monitored by fluorescence emissions, and the influences of exposure time and irradiation power were investigated. As shown in Figure 5D, the temperature of cell samples increased under microwave irradiation at different power. Heating efficiency was found to be proportional to the power of the irradiation. With irradiation power of 40 W and exposure time of 250 s, the


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temperature of the cell sample increased from 28 °C to 46 °C, which is sufficiently high to induce cell death.

Figure 6. Laser scanning confocal microscopy images of FTs labeled Hela cells at 23°C (A), 35°C (B) and 44°C (C). scale bars = 50µm. Microwave exposure-induced cellular temperature changes were also monitored using the confocal microscopy system. The ratiometrically changed fluorescence of the FT labeled cells was clearly observed and captured. As shown in Figure 6, under 488 nm laser excitation, the FT labeled cells exhibited fluorescence in both green (for R110) and red (for RB) channels, and the intensity of the red channel significantly decreased during the heating process. These results proved the feasibility of ratiometric R–F127–MF NP FTs in micro/nano thermometry applications. Conclusions


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We developed a three-step method to prepare a series of R–F127–MF NPs. In this process, the highly soluble dye molecules were associated with F127 micelles and incorporated in the crosslinked MF resin NPs during the high-temperature crosslinking treatment. Three kinds of nanoparticles (RB–F127–MF NPs, R110–F127–MF NPs, and R100–RB–F127–MF NPs) were prepared using this method, and R110–RB–F127–MF NPs were used for ratiometric fluorescence thermometry. The prepared dye-incorporated NPs are monodispersed with controllable size in the 80–110 nm range, bright fluorescence, and high stability during severe treatments, such as autoclave sterilization and long-term storage. R–F127–MF NP FTs exhibit high sensitivity of 15.4%·°C−1 and wide temperature sensing range of −20 °C to 110 °C. The FTs also exhibit excellent sensing accuracy in solutions with varied pH, ionic strengths, and viscosities, show unchanged working curve in cells, and provide the possibility of precise intracellular temperature measurement associated with diseases. With the prepared FTs, cellular temperature variations in Hela cells during the microwave exposures under different power and time were successfully monitored using a confocal microscope and proved the effectiveness of this FT in nanoscale thermometry applications in the biomedical field. ASSOCIATED CONTENT Supporting Information. Additional spectroscopic analysis and cell images. his material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]


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Author Contributions ‡These authors contributed equally. ACKNOWLEDGMENT This work was sponsored in part by the National Basic Research Program 973 of China (No, 2011CB707903), National Natural Science Foundation of China (81271686, 81228011, 61178085 and 81471771), the grants of Shaanxi province science and technology and innovation project (2011KTCL03-07). REFERENCES (1) Mecklenburg, M.; Hubbard, W. A.; White, E. R.; Dhall, R.; Cronin, S. B.; Aloni, S.; Regan, B. C. Nanoscale Temperature Mapping in Operating Microelectronic Devices. Science 2015, 347, 629-632. (2) Zijlstra, P.; Chon, J. W. M.; Gu, M. Five-Dimensional Optical Recording Mediated by Surface Plasmons in Gold Nanorods. Nature 2009, 459, 410-413. (3) Kucsko, G.; Maurer, P. C.; Yao, N. Y.; Kubo, M.; Noh, H. J.; Lo, P. K.; Park, H.; Lukin, M. D. Nanometre-Scale Thermometry in a Living Cell. Nature 2013, 500, 54-71. (4) Jaque, D.; Vetrone, F. Luminescence Nanothermometry. Nanoscale 2012, 4, 4301-4326. (5) Brites, C. D. S.; Lima, P. P.; Silva, N. J. O.; Millan, A.; Amaral, V. S.; Palacio, F.; Carlos, L. D. Thermometry at the Nanoscale. Nanoscale 2012, 4, 4799-4829. (6) Wang, X. D.; Wolfbeis, O. S.; Meier, R. J. Luminescent Probes and Sensors for Temperature. Chem. Soc. Rev. 2013, 42, 7834-7869. (7) Zhou, H.; Sharma, M.; Berezin, O.; Zuckerman, D.; Berezin, M. Y. Nanothermometry: From Microscopy to Thermal Treatments. ChemPhysChem 2016, 17, 27-36. (8) Sakaguchil, R.; Kiyonaka, S.; Mori, Y. Fluorescent Sensors Reveal Subcellular Thermal Changes. Curr. Opin. Biotechnol. 2015, 31, 57-64. (9) Neumann, P.; Jakobi, I.; Dolde, F.; Burk, C.; Reuter, R.; Waldherr, G.; Honert, J.; Wolf, T.; Brunner, A.; Shim, J. H.; Suter, D.; Sumiya, H.; Isoya, J.; Wrachtrup, J. High-Precision Nanoscale Temperature Sensing Using Single Defects in Diamond. Nano Lett. 2013, 13, 27382742. (10) Yang, J.-M.; Yang, H.; Lin, L. Quantum Dot Nano Thermometers Reveal Heterogeneous Local Thermogenesis in Living Cells. ACS Nano 2011, 5, 5067-5071. (11) Park, Y.; Koo, C.; Chen, H.-Y.; Han, A.; Son, D. H. Ratiometric Temperature Imaging Using Environment-Insensitive Luminescence of Mn-doped Core-shell Nanocrystals. Nanoscale 2013, 5, 4944-4950. (12) Albers, A. E.; Chan, E. M.; McBride, P. M.; Ajo-Franklin, C. M.; Cohen, B. E.; Helms, B. A. Dual-Emitting Quantum Dot/Quantum Rod-Based Nanothermometers with Enhanced Response and Sensitivity in Live Cells. J. Am. Chem. Soc. 2012, 134, 9565-9568.


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Table of Contents or Graphical Abstract


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Ratiometric Nanothermometer Based on Rhodamine Dye

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