Nanothermometry for cellular temperature monitoring and disease diagnostics

Body temperature variations, including the generation, transfer, and dissipation of heat, play an important role throughout life and participate in all biological events. Cellular temperature information is an indispensable link in the comprehensive understanding of life science processes, but traditional testing strategies cannot provide sufficient information due to their low precision and inefficient cellular‐entrance. In recent years, with the help of luminescent nanomaterials, a variety of new thermometers have been developed to achieve real‐time temperature measurement at the micro/nano scale. In this review, we summarized the latest advances in several nanoparticles for cellular temperature detection and their related applications in revealing cell metabolism and disease diagnosis. Furthermore, this review proposed a few challenges for the nano‐thermometry, expecting to spark novel thought to push forward its preclinical and translational uses.


| INTRODUCTION
Temperature plays an important role during the lifecycle, which not only affects the lifestyle and life activities of organisms but also reflects their health state.Large-scale animal migration, 1 plant growth and development, 2 microbial community composition and metabolism 3 are always regulated by seasonal changes in temperature, and each system is in a state of dynamic equilibrium. 4The body's powerful thermoregulatory system maintains body temperature constant, which is mainly controlled by the central nervous system.The ambient temperature is first perceived through cutaneous thermoreceptors, then this information is routed to the brain via spinal cord and midbrain, and integrated into the hypothalamus. 5If the core temperature of the human body is high, the anterior hypothalamus will receive information to drive cooling behaviors such as sweating; conversely, low core temperature transmission to the posterior hypothalamus reduces sweating or stops driving warming behavior. 6In addition to maintaining a constant core body temperature against changes in the external environment, thermoregulation can reflect the occurrence and development of diseases to a certain extent. 7Fever is a complex adaptive response of the host to numerous immune challenges. 8It is also a pathophysiological response and an important vital sign in clinical diagnosis and treatment. 9ever is always accompanied by the occurrence of various diseases such as infectious, 10 inflammation, 11 allergies, 12 etc.For example, for highly contagious infectious diseases such as avian influenza and COVID-19, fever is a predictor of adverse consequences, so it is necessary to continuously monitor body temperature at strict intervals. 13,14Patients in Intensive Care Unit have a high risk of disease aggravation caused by infection, and body temperature monitoring is often used as one of the criteria to judge whether the symptoms have worsened. 9oreover, sustained high core temperature is lifethreatening.When people are exposed to high temperature and humidity for a long time, it is easy to cause heat exhaustion, and even lead to heat cramp and heat stroke. 15Once the core body temperature exceeds 40 ˚C, it is clinically recognized as hyperthermia.In severe cases, it is accompanied by the risk of symptoms such as bleeding and coagulation disorders, circulatory failure, systemic inflammatory response, and multiple organ dysfunction syndrome. 16Therefore, core body temperature is inseparable from human life activities and is also an important indicator in the process of disease development and treatment.
Cells are the basic units and participate in all activities of living organisms. 17The human body is composed of more than 30 trillion cells. 183][24][25] Under cold stimulation, brown adipocytes produce heat via mitochondrial uncoupling protein 1 (UCP1). 26In general, protons are output from the mitochondrial electron transport system, creating an energy gradient across the inner mitochondrial membrane and providing thermodynamically favorable free energy for adenosine triphosphate (ATP) synthesis. 26evertheless, UCP1, located on the inner mitochondrial membrane, can loot the energetic gradient in the inner mitochondrial membrane and generate heat by pulling protons from the electron transport chain away from ATP production. 26When cells experience heat stress, high temperature causes protein unfolding, tangling and unspecific aggregation, resulting in the imbalance of protein homeostasis in cells, which eventually leads to a change in cell morphology and a decrease in cell function. 27,28For abnormal cells, such as tumor cells, the energy metabolism is significantly faster than that of normal cells. 29The abnormal energy metabolism of tumor cells is highly dependent on aerobic glycolysis, which can quickly release more energy in the cytoplasm and increase the tumor temperature significantly. 30Therefore, cell temperature monitoring and intracellular heat distribution maps not only help to explain cellular events but also provide the possibility to discover diseases at the cellular level and develop new diagnostic and therapeutic methods.
Although cell temperature is very important for life activities, universal detection of cell temperature is not possible with current technology.For body temperature detection, a variety of new temperature measuring instruments have been developed in the existing technology, such as large infrared thermal imaging cameras, 31 wearable health monitoring equipment, 32 electronic thermometers, etc..However, these devices are not suitable for high-precision and high-sensitivity measurements at the cellular level.First of all, the size of cells is in the range of nano/micron meters. 33nly thermometers as small as nanometers can sufficiently contact the temperature environment inside and outside the cells.Secondly, living cell environments are complex, and temperature mapping needs high temporal and spatial resolution. 34Finally, the temperature measurement of cells requires good biocompatibility and low toxicity. 35In recent years, the vigorous development of optical technology and nanomaterials has made it possible to establish varieties of temperature sensing methods in living cells using fluorescence microscopy, while making accurate and sensitive temperature sensing of cells in vitro and in vivo. 36Among them, nanomaterials with unique optical and temperature-sensitive properties have been used as nanothermometers to measure cell temperature, such as Quantum dots (QDs), 37 rare earth nanoparticles, 38 metal nanoparticles, 39 carbon nanomaterials, 40 and polymer nanospheres.Therefore, this article summarizes the properties of various nano-thermometers and their applications in intracellular and extracellular temperature monitoring.At the same time, nanothermometers used for the investigation of organelles, cellular physiological processes, and disease diagnosis are also discussed (Scheme 1).

CELLULAR TEMPERATURES
Nano-thermometry combines with spectroscopic technique to obtain luminescence characteristics of the probes as temperature indicators. 41The four typical thermos-sensing strategies are (a) luminescence intensity, (b) luminescence intensity ratio, (c) peak position and (d) fluorescence lifetime (Figure 1).3][44][45] At high temperatures, the internal electrons of nanoparticles are activated and the internal energy is redistributed.It is always accompanied by the escape of charge carriers from luminescence center of nanoparticles, the intensification of deformation and vibration of the internal structure of nanoparticles, and the increase in the number of collisions of nanoparticles, which greatly consumes non-radiative energy and leads to the change of luminescence performance of nanoparticles.In detail, the temperature detection strategy based on luminescence intensity mainly includes emission intensity variations or intensity ratio changes between two different emission peaks.The emission intensity always gradually decreases with increasing temperature.In the case of intensity ratio detection strategy, the temperature sensitivity of two emission centers of the probes is not uniform, leading to an intensity ratio changes with improving detection reliability. 46It has been reported that thermal induction greatly enhances the nonradiative energy consumption, causing a change in the emission energy level and thus a redshift in the emission peak.The reason for lifetime as a temperature indicator lies in the fact that the increase in ambient temperature accelerates nonradiative processes, so that the time to complete the whole luminescent process becomes shorter relative to a low temperature environment. 34ifferent strategies were used to detect intracellular and extracellular temperature changes depending on the luminescence characteristics of various nanoparticles.

| Quantum dots-based nanothermometers
Quantum dots have great potential in biological applications due to their excellent photostability, high brightness, small size and easy surface modification. 47attice dilation and electron-phonon coupling are the main contributors to temperature variation in the QD energy gap. 43When temperature increases, the excitonic emission from QDs is increased and the lattice of QDs is thermally expanded, resulting in the variation of both photoluminescence intensity and emission position. 48herefore, QDs usually establish a relationship with temperature through luminescence intensity or luminescence peak position.
0][51][52] In terms of polymermodified QDs, Wu et al. reported that the QD-micelle composite could be efficiently absorbed by cells and evenly spread in the cytoplasm. 53CuInS 2 /ZnS QDs micelles (QDs-micelles) were obtained through encapsulating CuInS 2 /ZnS QDs by polyethylene glycol derivative (poly-oxyethylene stearate).The fluorescence intensity of QDs-micelles gradually reduced with an increase in ambient temperature, which was proven to have hightemperature sensitivity (2.0% ˚C−1 ) in phosphate buffer solution and independent environmental factors such as pH, ion concentration, and protein content.By observing the distribution of QD-micelles in cells, it was found that most QD-micelles could be evenly distributed in the cytoplasm, while a few QD-micelles appeared to aggregate into bright spots in some cells.It was proved that intracellular temperature measurement was not influenced by the aggregation state of QD-micelles, ensuring the accuracy of temperature sensing.As the temperature gradually increased, the fluorescence intensity of QD-micelles decreased significantly, and the linear relationship between fluorescence intensity and temperature changes was obtained through statistical methods.In addition, doping an appropriate amount of Cu 2þ or Mn 2þ in QDs can obtain intrinsic dual-emission QDs, whose ratio can be adjusted by changing dopant elements.These transition metal ion-doped QDs provide a promising tool for ratiometric optical thermometric sensing. 52,54ang et al. combined QDs and double helix-point spread function (DH-PSF) technology to synchronize spatial positioning with temperature readout, enabling 3D targeted thermal imaging of tumor cells at the subcellular level. 37Water-soluble CdTe QDs were prepared with the aqueous method, and transferrin (Tf) was modified on the surface of CdTe QDs.Tf could specifically bind to transferrin receptor overexpressed on the surface of tumor cell membranes to achieve tumor cell targeting (Figure 2A).The emission wavelength of Tf-QDs gradually red-shifts with increasing temperature in a linear relationship, and its temperature sensitivity was 0.15 nm ˚C−1 .A threedimensional nanoscale tumor cell thermal imaging system was constructed by incorporating the relationship between the axial defocusing distance and rotation angle of the double light spot in DH-PSF technology to gain the spatial position and temperature data of the sensors in tumor cells, which provided support for microscopic inhomogeneity of the temperature section on the surface of cancer cells (Figure 2B).

| Lanthanide-based luminescent nanothermometers
6][57][58] Lanthanide luminescent nanomaterials utilize the sensitizer and activator of lanthanide ions to absorb photon energy and convert it into luminescence. 59,60The temperature sensitivity of lanthanide luminescent nanoparticles is shown by the thermal response distribution of excited states, energy transfer between lanthanide ions, or between lanthanide ions and phonons. 61The temperature-dependent emission for a single type of lanthanide ion conforms to the Boltzmann statistical distribution theory.The electron distribution of two adjacent excited state levels is connected with temperature, resulting in changes in emission intensities. 61Since phonons are controlled by temperature, the mutual energy transfer between different species of lanthanide ions with the assistance of phonons is also affected by temperature.For example, the sensitizer ion acquires energy and transfers it to the emitter under excitation.In turn, the excitation energy can be transferred back to the sensitizer with the participation of lattice vibrations (phonons). 62In addition, phonons can relocate the energy of the harvested excited lanthanides through a nonradiative relaxation process (namely phonon relaxation), thereby altering the emission. 61For thermometry, the luminescence lifetime and intensity ratio of lanthanide nanoparticles have their own characteristics.The luminescence lifetime, as an inherent characteristic of lanthanide ions, can be ignored by biological tissue, which can improve the accuracy of reading. 57The intensity ratio detection is not affected by the concentration of fluorophore and excitation source, which can be performed by self-calibration to improve detection resolution. 63anthanide luminescent nanomaterials are divided into down-conversion and upconversion luminescence. 60,64There are many applications of downconversion luminescence nanoparticles in in vivo temperature detection.For example, Jacinto et al. designed the co-doped Yb 3þ /Nd 3þ core/shell nanoparticles to measure subcutaneous thermal transients in small animal models to obtain fundamental properties such as absorption coefficient and thermal diffusivity of the relevant tissues. 64Jaque et al. synthesized downconverted luminescent nanoparticles capable of in vivo photothermal heating, fluorescent tumor localization, and intratumoral thermal sensing, emphasizing the importance of temperature measurements to control therapeutic parameters, which are not achievable by conventional body temperature measurements. 65ue to the unique luminescence process of converting low-energy excitation into high-energy emission, upconversion luminescent nanoparticles (UCNPs) have been used in considerable research in cell thermometry. 66Yang et al. designed lanthanide-doped upconversion luminescence nanothermometers for accurate temperature mapping on immune cell membranes for the first time. 38First, due to the thermal coupling energy level of Er 3þ , they synthesized core-shell NaGdF 4 : Yb(8%)/Er(1%)@NaGdF 4 UCNPs for ratio upconversion luminescence thermometry (Figure 3A).At 980 nm excitation, core-shell UCNPs showed two narrow green emission peaks at 525 nm ( 2 H 11/ 2 → 4 I 15/2 ) and 545 nm ( 4 S 3/2 → 4 I 15/2 ) (Figure 3A).When the temperature increases from 293 to 353 K, the luminescence intensity ratio at 525 and 545 nm (ie, I 525 /I 545 ) increases by 1.6 times with a relative sensing sensitivity of 1.4%/K.Next, dibenzocyclooctane was modified on the surface of UCNPs (UCNPs-DBCO) to target the azo group on the cell membrane via metabolic glycoengineering, so that UCNPs could be localized on immune cell surface (Figure 3B).Finally, upconversion hyperspectral imaging technology was used to record upconversion luminescence signals of UCNPs-DBCO on cell membranes at different temperatures, and the standard curve was obtained.At the same time, thermally induced Ca 2þ influx is also illustrated in Figure 3C.Stromal interaction molecular-1 (STIM1) was activated and accumulated at the endoplasmic reticulumplasma membrane junctional sites in response to thermal stimulation, modulating the Ca 2þ -permeable ORAI1 ion channel.The fluorescence intensity was significantly enhanced with the increase in temperature, indicating that UCNPs-DBCO could accurately reflect the change of cell temperature.The results also indicated that there was a positive correlation between Ca 2þ influx and temperature in CD69 cells.It was also found that although the intensity of single-peak signal per pixel varied greatly, the cell membrane temperature calculated only fluctuated mildly along the cell membrane by analyzing signal ratiometric.
In addition, the luminescence ratio probes were used to measure temperature variations by using Er-doped UCNPs. 68For example, Li et al. combined silica-modified UCNPs with negatively charged natural protein spider silk to detect tumor cell temperature. 67The UCNPdecorated spider silks can approach C127 cell membranes on the glass slide via six-axial micro platforms until the silk adheres to the membrane (Figure 3D).A 980 nm laser beam was coupled into spider silk to excite UCNPs, as shown through the dark-area image (inset of Figure 3E).The fluorescence spectra were obtained at the membrane near the cell (R1) and an extracellular point near the cell (R2).The fluorescence intensity ratios of R1 and R2 positions were 0.291 and 0.246, respectively.Based on the dependence of the obtained temperature change on the fluorescence intensity ratio, the temperatures at the R1 and R2 positions were calculated to be 313 and 298 K, respectively.This result indicated that the nearer to the nucleus, the higher the temperature of the cell membrane (Figure 3F).

| Metal nanoclusters-based nanothermometers
Metal clusters, composed of several to dozens of metal atoms, have a size comparable to the electron Fermi wavelength and exhibit some molecular-like properties, such as discrete electronic states and strong sizedependent fluorescence in ultraviolet to infrared regions. 69,70Therefore, metal clusters have received widespread attention in sensing and imaging.Research on intracellular temperature sensing mainly focused on gold nanoclusters (AuNCs) and copper nanoclusters (CuNCs).
The luminescence properties of AuNCs are temperatureresponsive.The increasing temperature induces quenching of luminescence intensity and reduction of lifetime in the case of AuNCs.The reasons for this phenomenon are classified as follows.(1) High temperature can weaken the Au-S bond between the ligand and Au core, resulting in reduced charge transfer from the ligand to the Au core, triggering fluorescence quenching.(2) High temperature causes an increase in non-radiative transition rate and molecular collision frequency, which is attributed to the enhancement of non-radiative decay. 71Nienhaus et al. used lipoic acid-modified AuNCs as fluorescent probes. 72he fluorescence lifetime of AuNCs changed significantly within the physiological temperature range.The lifetime variations of AuNCs in the cellular environment were much greater than that in phosphate buffered saline, making them very suitable for intracellular temperature sensing.It can be seen that the fluorescence lifetime of AuNCs after endocytosed by HeLa cells was gradually shortened with increasing temperature (Figure 4A).
In addition to intracellular temperature testing, suborganelle temperature sensing can be performed.Reproduced with permission. 67Copyright 2021, American Chemical Society.
AuNCs with long fluorescence lifetimes were modified with the 4-(carboxybutyl) triphenyl phosphonium bromide, which can be used as a highly sensitive and stable fluorescence lifetime thermometer for the temperature of intracellular mitochondria (Figure 4B). 73he surface of TPP-modified AuNCs had negative charges, which could target mitochondria.In this way, the modified AuNCs had a microsecond-level fluorescence lifetime, which gradually decreased with the increase in temperature.The relative sensitivity of AuNCs was as high as 2.8%, and they had a high resistance to photobleaching.It can be seen from Figure 4C that the temperatures of region of interest (ROI) were different in the single cell.The fluorescence lifetime of mAuNCs in ROI b was longer than that in ROI a, which implied that ROI a was at a higher temperature.These results demonstrated the heterogeneity of mitochondrial temperature in individual cells.Compared with the precious metal Au, Cu was abundant and cheap on the earth, and CuNCs also had temperature-sensitive photoluminescence properties.A novel fluorescent CuNCs was reported by Song et al. using glutathione as both the reducing agent and protective layer by one-step method. 74The synthesized GSH-CuNCs showed outstanding biocompatibility and sensitivity to temperature, and the imaging of MC3T3-E1 cells at different temperatures can be seen through confocal imaging.

| Carbon nanomaterials-based nanothermometers
Due to their excellent optical, thermal, electronic and mechanical properties, carbon nanomaterials are widely used in many fields such as energy production and storage, materials science, biology and medicine. 75,76hrough continuous exploration, carbon nanomaterials have been named as various types of nanoparticles, such as graphene, carbon nanotubes, carbon nanofibers, Carbon dots (CDs), carbon nanodiamonds, etc. 77 Many carbon nanomaterials have excellent photoluminescence properties, easy functionalization, and large surface areas, which make nanoplatforms with imaging functions, good biocompatibility and drug loading. 78arbon dots are 0-dimensional carbon-based nanomaterials with an average diameter of less than 10 nm, composed of sp 2 /sp 3 carbon skeleton and rich functional groups. 79CDs have the advantages of simple preparation, low cost, multiple luminescence mechanisms, and tunable optical properties. 80The sensitivity of CDs to temperature is manifested in the decrease of luminescence intensity with temperature, which may be caused by the enhancement of thermally activated non-radiative channels in the surface (trap) state. 81At lower temperatures, radiative recombination plays a leading role which results from the enhancement of emission intensity due to robust electronhole interactions.Correspondingly, higher temperatures favor nonradiative recombination in which electronphonon interactions are more salient than electron-hole interactions. 81Xiao et al. reported an intracellular temperature-sensing nanothermometer based on redemitting carbon nanodots (RCDs). 82RCDs with outstanding fluorescence properties were synthesized by the solvothermal method using thionine and citric acid as raw materials, which had a good linear relationship, reversibility and reproducibility between temperature and fluorescence in the range of 4-80 ˚C (Figure 5A).Wang et al. used a facile annealing to prepare dual emission nitrogen-doped CDs (N-CDs) by solvent-free carbonization of ammonium citrate in air. 83N-CDs appeared dual emission peaks at 462 and 560 nm under 360 and 470 nm excitation, respectively.When the temperature increased, the nonradiative channel was thermally activated and the emission of excitons was reduced due to the presence of defects on the surface of CDs, resulting in the decrease of fluorescence intensity.As shown in the Figure 5B,  73 Copyright 2021, American Chemical Society.
different excitation wavelengths of N-CDs exhibited multimodality for the sensing of cell temperature.
Nanodiamond is another platform for fluorescence thermometry of carbon nanomaterials, which can respond to temperature by utilizing the temperature variation properties of the nitrogen-vacancy (NV) color center energy spectrum. 85NV color centers are point defects in diamond, which consist of a substitutional nitrogen atom next to a carbon vacancy. 85The ground and excited states of the NV-color center spin quantum states are a spin tristate, resulting in a resonant spin transition at 2.87 GHz and an optical transition with zero phonon line (ZPL) at 637 nm. 86This means that a rise in temperature causes the NV − center to increase the resonance frequency of the spin transition at 2.87 GHz or the ZPL of electronic transition at 637 nm to produce a thermal shift.Therefore, the temperature measurement can be achieved by using the resonance frequencies of optically detected magnetic resonance and spectral shift of the ZPL emission wavelength. 87Li et al. combined fluorescent nanodiamonds (FNDs) with a scanning optical tweezer system to study cell temperature and precise intracellular localization. 84As shown in Figure 5C, the dispersed FNDs were uptaken by living cells and then trapped by an optical trap (upper).Microspheres were gradually assembled by FNDs with the sustained influence of the trapping laser (lower).The luminescence intensity of FNDs at 546 nm was increased by a factor of seven, which was caused by the formation of FNDs microsphere and electromagnetic resonance between individual nanodiamonds.With the help of scanning optical tweezers system, the temperature at diverse locations in human brain microvascular endothelial cells can be mapped by taking the fluorescence spectrum of the microspheres (Figure 5D).In this way, temperature changes can be precisely detected at any location or organelle in a living cell.In addition to NV − center, Weil et al. explored FNDs with silicon-vacancy centers (SiV) through the high pressure and high temperature based on metal-free catalyst growth. 88The NIR emission of FND-SiV allows deeper tissue penetration and in vivo imaging compared to FND-NV centers.With the modification of polymers, FNDs with SiV can achieve dual-color imaging of live cells and intracellular tracing.In a word, NDs can provide a new way for the application of live cell biological imaging, diagnosis and treatment.

| Polymer-based nanothermometers
Polymer nanothermometers with unique properties are another important tool for intracellular temperature sensing and imaging.In order to achieve rapid and visual temperature sensing by polymer nanothermometers, it is often necessary to combine polymers and fluorescent luminescent groups with indicative effects. 89The principle of a thermosensitive polymer nanothermometer could be generalized as follows: the phase change of thermosensitive polymers can be converted into the signal of fluorescent dyes, leading to their optical or emission characteristic changed along with ambient temperature. 89,90When the temperature is below the lower critical solution temperature (LCST), the fluorescent polymers exist in a coil state; at the temperature above the LCST, the polymers react to the local ambient temperature and their phase changes from coil to spherical. 91,92Meanwhile, the signal transmission of fluorescent units can be observed as an indicator section.A large number of synthetic and natural polymers display thermos-responsive behaviors, such as poly[oligo(ethylene glycol) methacrylate] (POEGMA), poly(N-diethylacrylamide) (PDEAm) and poly(N-isopropylacrylamide) (PNIPAm). 93PNIPAm is the most discussed thermosensitive polymer material with LCST of 32 ˚C.PNIPAm can dissolve in water and has an extended conformation below LCST, while above LCST it transforms into hydrophobic spheres to form hydrogel. 94 Zhang et al. reported an ultrastable color-tunable fluorescent hydrogel based on PNIPAm as intracellular thermometer. 95As shown in Figure 6A, three polymers were obtained via atom transfer radical polymerization, including rhodamine B-based N-isopropylacrylamide polymer (RNP), the fluorescein-based N-isopropylacrylamide polymer (FNP), and the tetraphenylethylene (TPE)-cage-based N-isopropylacrylamide polymer (CNP) with red, green, and blue fluorescence, respectively.Since TPE was a classic aggregation-induced emission molecule, the fluorescence intensity of CNP (λ = 450 nm) obviously increased at around LSCT and showed a mildly decreasing trend with increasing temperature.On the contrary, rhodamine B and fluorescein are conventional chromophores with aggregation had quenching effects, which were involved in the formation of polymers whose fluorescence intensity decreased gradually with an increase in temperature.Due to the inconsistent changes in the sensitivity of CNP, FNP, and RNP fluorescence intensities to temperature, tuning the mass ratio of these polymers could result in full-color-tunable fluorescent nanohydrogels, including nanohydrogels with white-light emission (NWLEs) (Figure 6B).When the NWLE underwent heating-cooling cycles of 25-50 ˚C, the fluorescence spectra exhibited excellent reversibility, and the fluorescent color of NWLE was consistent with the Commission internationale de l'éclairage coordinate plot (Figure 6C).After incubation of HeLa cells with NWLE for 4 h, fluorescent variation at temperatures 25, 33, 37 and 42 ˚C were studied with laser confocal microscopy.As the temperature increased from 25 to 42 ˚C, fluorescence color turned from white to blue and then orange.These results suggested that NWLE was an ideal, ultra-stable material for cellular thermometry.
Non-thermosensitive polymer nanothermometers include structurally conjugated polymers (e.g., polydiacetylene) and fluorescent dyes physically entrapped in polymers. 89The temperature measurement mechanism of structurally conjugated polymers is due to the stressinduced nonfluorescence-to-fluorescence transition properties of conjugated fluorescent polymers, which causes the fluorescence intensity of luminescent units responsive to temperature enhancement. 97,98The thermometry principle of fluorescent dyes physically entrapped in polymers is that the polymer serves as a matrix to inhibit vibration and rotation of the fluorescent group, thereby causing the fluorescence intensity to change with temperature. 99,100Tian et al. used non-thermosensitive polymer F127 as the matrix and encapsulated two fluorophores 2-([1,1 0 -biphenyl]-4-yl)-3-(4-((E)-4-(diphenylamino)styryl) phenyl) fumaronitrile (TBB) and the reference dye Rhodamine 110 to establish the ratiometric fluorescence thermometer, TBB&R110@F127 nanoparticles (Figure 6D). 96Over the temperature ranges of 25-65 ˚C response, the TRF NPs exhibited excellent temperature sensitivity (2.37% ˚C−1 ) and outstanding temperature-sensitive fluorescence reversibility (Figure 6E,F).Intracellular thermometry experiments showed that TRF NPs could monitor the temperature rise of Hep-G2 cells from 25 ˚C to 53 ˚C during the heating of photothermal therapeutic agents, demonstrating that TRF NPs had considerable potential applications in the field of biological thermometry.
We further summarized various nano-thermometers in Table 1, in which different nanoparticles were used, including the temperature response signal, sensitivity, and sensing cell types.Fluorescence measurement is a commonly used detection method at the cell level, in which detection signals include luminescence intensity, ratio luminescence, fluorescence lifetime and emission peak location.The range of cell temperature examined is mostly 25-45 ˚C, and it mainly detects the temperature of cell periphery, cell membrane, cell interior and targeted mitochondria and other organelles.These nanoparticles  can be used for multiple-cell temperature detection and more accurate single-cell temperature detection based on their unique temperature sensitivity and inertness to the complex cellular environment.It provides a basis for the realization of temperature sensing in vivo and disease diagnosis and treatment.

| APPLICATIONS
3.1 | Response to the cellular physiological state Cell metabolism is an important process of cell life activities, and intracellular temperature maintenance is in a dynamic range during this process.Real-time intracellular temperature imaging opens new ways to understand in detail the endogenous or exogenous stimuliinduced thermal gradients in living cells, offering valuable insights into the mechanisms of intracellular regulation and facilitating a better understanding of the functions of some heat-generating organelles in the cellular functions. 104In recent years, researchers have begun to use micro/nanoscale thermometers with high resolution and accuracy to explore intracellular heat changes in living cells.Carlos et al. used Ln 3þ -doped polymer micelle probes to test the temperature of breast cancer cells in medium from 296.45 to 303.55 K. 105 The results in Figure 7A,B indicated that the temperature was unevenly distributed within the cell, which was associated with the activity of the thermo-generating organelles and highly exoenergetic processes.
As an important place for cellular energy conversion, various metabolic substrates are converted by mitochondria into two forms of energy: ATP and heat. 106Qi et al. simultaneously detected temperature and ATP during oxidative phosphorylation in living cells by synthesizing a temperature-sensitive probe and an ATPsensitive probe targeting mitochondria. 107It was demonstrated that the temperature in mitochondria increased by 2.4 ˚C and the fluctuation level of ATP reduced by 75% within 2 min during the oxidative phosphorylation process.Jin et al. obtained upconversion nanothermometers that efficiently targeted mitochondria to study mitochondrial thermogenesis in situ under different stimulation conditions. 102They have found that F I G U R E 7 (A) Microscopy images of the MDA-MB-468 cells incubated with DNPD-based polymeric micelles.The blue and red points mark of the locations used for temperature determination of the darker and brighter regions, respectively.(B) Temperature histograms obtained from the selected points indicated in (A).Reproduced with permission. 105Copyright 2020, American Chemical Society.Visualization of mitochondrial thermal dynamics in HeLa cells response to nutrient and chemical stimulations (C) left: 5 mg/mL glucose; right: 5 μM oleic acid; (D) left: 1 μM ionomycin calcium salt; right: 10 μM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (The left, middle and right panels of the combined images are UCNP@TPP images, mitochondrial temperature dynamics and the test of results with and without stimuli).Reproduced with permission. 102Copyright 2021, American Chemical Society.(E) Distinct lysosomal and mitochondrial temperature dynamics in response to chemical stimulations in living HeLa cells (left: Ca 2þ ion shock and dimethyl sulfoxide (control); middle: chloroquine (CQ) and PBS (control); right: FCCP and DMSO (control).Reproduced with permission. 103Copyright 2022, PNAS.PBS, phosphate buffered saline.

LIU ET AL.
HeLa cells cultured with high glucose and lipid nutrient conditions have different mitochondrial thermogenesis responses (Figure 7C).Compared with high-glucose medium, mitochondria cultured in lipid medium reacted faster and maintained relatively high temperatures for a longer period of time.As displayed in Figure 7D, both Ca 2þ promoting ion pumping and oxidative phosphorylation inhibiting enzymes destroying ATP synthesis could force cells to release heat.This result indicated that mitochondria exhibited different thermodynamic processes in different culture environments, highlighting the wide application of nanothermometers in the study of vital biological processes of mitochondrial metabolism as well as interactions among mitochondria and other organelles.Lysosomes and mitochondria are two of the most metabolically active cellular organelles, and mutual regulation of energy between them is of great significance to cellular homeostasis. 108Jin's research group also synthesized temperature-sensitive LysoDots and MitoDots to target lysosomes and mitochondria, respectively. 103They stimulated HeLa cells with Ca 2þ , chloroquine (CQ) and carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) respectively, and used the fluorescence ratio changes of LysoDots and MitoDots to observe the thermodynamic changes between lysosomes and mitochondria.In this work, they have observed intriguing spatial and temporal temperature dynamics in HeLa cells.Under the shock of organelle nonspecific Ca 2þ ions, the temperature of lysosomes and mitochondria rose by 2-4 ˚C (Figure 7E, left).With the lysosome-specific drugs CQ treatment, the temperature of lysosomes decreased by about 3 ˚C, while the temperature of mitochondria remained 37 ˚C (Figure 7E, middle).With the mitochondria-specific drug FCCP treatment, the temperatures of lysosomes and mitochondria both jumped by 3-7 ˚C (Figure 7E, right).These observations suggested the existence of distinct metabolic pathways and thermal conversion between lysosomes and mitochondria.

| Diseases diagnosis and treatment
Body temperature is an important parameter in judging the vital signs of patients, which is closely related to the diagnosis and treatment. 9In the development of bacterial infection, viral infection, inflammation and other diseases, high core body temperature (fever) is usually shown, which is the body activating its own autoimmune protective mechanism.Fever can occur in any part of the human body.For example, the brain temperature of healthy people is between 36.1 and 40.9˚C, while the brain temperature of patients with traumatic brain injury ranges from 32.6-42.3˚C. 109Daily rhythmicity of brain temperature in patients with traumatic brain injury emerges as one of the strongest single predictors of survival after brain injury.The core temperature of patients with heat stroke ranges from 40 to 44.8 ˚C, accompanied by severe loss of consciousness. 110Without timely cooling treatment, it can induce early multi-organ damage, rapidly progress to organ failure, and eventually lead to death.For viral infections, an increase in physiological temperature of only 1.5 ˚C from 36.5 ˚C to 38 ˚C strongly increases the antiviral immune response. 111Since the elderly have lower body temperature than children and young people, the expression of antiviral genes in the elderly is reduced, and the elderly are more threatened by viral infection. 111In patients with serious sepsis, hyperthermia and hypothermia have different effects on mortality and severity of physiological function decline. 112Among patients, body temperature below 36.5 ˚C was dramatically related to an elevated risk of death, more than twice as high as in patients with nonhypothermia.In addition, a variety of diseases such as tumors, stroke, neurodegenerative diseases, sepsis and so on show fever symptoms, so it is very important to detect fever in the development of various diseases. 113,114owever, only a few of these diseases have studied the temperature changes at the tissue and cell level.Most of them still focus on the detection of core temperature and have not yet studied in depth.For nanothermometers, deep fever research has been carried out on cancer and ischemic stroke.

| Cancer
Fever dominates the clinical symptoms of tumors, and can be explored as an indicator for diagnosis and treatment of tumors to some extent. 115Cancer-associated fevers can be divided into three categories: (1) cancer-associated infectious fevers 116 ; (2) fever caused by the development of tumor itself, including the release of endogenous thermogenic factors by tumor cells, thermogenesis during rapid tissue proliferation, and thermogenic sources released by immune response 117 ; (3) long-term excessive body consumption in cancer patients leads to a fever caused by the imbalance of thermoregulatory center.In addition, the corresponding thermo-variations in tumor tissues can also reflect the high metabolic activity of tumor growth as well as the changes in blood perfusion caused by new tumor vasculature. 118,119hermosensitive nanothermometers can be used to measure intra-tumor temperature and assist in the diagnosis and treatment of cancers.Fernández et al. monitored changes in tumor thermal relaxation dynamics using an Ag 2 S infrared luminescence nanothermometer (transient nanothermometer, TTh) and studied the evolution of thermodynamics throughout the entire growth process of melanoma mouse model from induction to the final stage of obvious necrosis. 120In this work, Ag 2 S-PEG and tumor cells were injected into mice at the same time, and they summarized and compared the relationship between thermal relaxation dynamics, tumor volume, and temperature between the healthy tissue and the tumors with time (Figure 8A).It was found that TTh could detect the presence of tumors only 5 days after inoculation of cancer cells, which is 6 days earlier than traditional diagnosis based on optical inspection.Blood perfusion increased in the early stages of tumor development, and then gradually decreased in the later stages.This is because high metabolism generates a vascular network in the early stage of tumor development; However, in the later stages, the blood flow of tumor is drastically reduced by the extravascular high pressure acting on the blood vessels, resulting in the inefficient absorption of oxygen and nutrients, which promotes tumor internal necrosis.By comparing blood perfusion and thermal relaxation times, TTh can be used to identify early and late stages of tumor development.
Nanothermometers can assist in the treatment of tumors with hyperthermia or drug release.Photothermal therapy (PTT) usually maintains the temperature of diseased area at 42-45 ˚C or even higher using high temperatures to kill cancer cells. 101,122Due to the large temperature difference in vivo and in vitro during PTT, excessive PTT conduction can increase the damage of normal tissues adjacent to the lesion, and insufficient PTT will lead to a prolonged treatment period. 123In order to increase the accuracy of hyperthermia, internal temperature sensing is very important.In view of this, Daniel's team proposed PbS/CdS/ ZnS QDs PTT based on temperature self-monitoring. 121hese QDs simultaneously served as photothermal agent and high-resolution fluorescent thermal sensors (Figure 8B), making it possible to completely control the elevated temperature of the tumor during PTT.As shown in Figure 8C, the power density of the excitation was controlled through the temperature range required of PTT, and it is concluded that the optimal treatment temperature could be obtained by adjusting the power within the range of 1.5-2.0W cm −2 .The surface burns after PTT treatment healed completely after 3 weeks, leaving only a surface scar (Figure 8D).It can also be seen in Figure 8E that PTT treatment was performed in the optimal power range and the mass volume was completely ablated.In this study, differences in intratumoral and surface temperatures under different irradiation conditions were observed, emphasizing the real-time control of tumor temperature to dynamically adjust the treatment conditions to maximize the therapeutic effect.

| Ischemic diseases
In ischemic diseases, the temperature of the ischemic site fluctuates greatly due to ischemic injury and inflammatory response.Ischemic diseases occur when blood vessels are constrictive or occluded under various pathological conditions, manifesting as a sudden interruption of blood supply leading to temporary or permanent hypoxia and nutrient deficiency. 124The extent and duration of ischemic injury are closely related to the severity of cell death and tissue damage.Myocardial infarction and stroke are the most prominent examples of ischemic diseases. 125Transient ischemic attack with resolution of symptoms soon after the initial event may also result in inflammation of vascular endothelial cells, lacunar infarction, and lead to further induced thrombosis. 126If left undiagnosed and untreated, ischemic stroke can give rise to permanent brain damage, prolonged disability, and even death.
The general methods for detecting cerebral ischemia mainly focus on CT and MRI imaging, but these two imaging methods have problems such as long detection time or poor sensitivity and specificity. 127,128Zhu et al. solved this problem using temperature-responsive NIR emitting lanthanide luminescent nanoparticles. 129Brain temperatures are higher than in unaffected areas and the body due to the ischemic metabolic response, the impairment of brain heat exchange function caused by changes in cerebral blood flow, and the activity of early inflammatory cells.Therefore, the temperature in the brain can be used as an indicator of ischemic stroke for early diagnosis and disease tracking.In this work, the PEG-modified NIR emissive NaNdF 4 :Yb@CaF 2 @-NaNdF 4 :Y (NYCN@PEG) nanoparticles showed excellent temperature sensitivity in the range of 10-70 ˚C and exhibited a strong luminescence signal in a transient middle cerebral artery occlusion model.When NYCN@PEG was injected intravenously 2 h after reperfusion, a progressive increase in the intensity was observed from 3 to 24 h (Figure 9B), suggesting worsening of ischemic brain damage.As shown in Figure 9C, the NIR-II luminescent signal (green) was located in the core area of the infarct, which overlapped with the area of massive apoptotic and necrotic cells (red), and no nanomaterials were detected outside the ischemic region.It was also found that the temperature at both time points (Figure 9D) was higher than the liver temperature, suggesting a significant difference between the temperature in the ischemic region and the core body temperature (liver).This result indicated that the temperature of ischemic region was associated with the advancement of the pathological state, and the brain temperature at the ischemic site can be taken as a marker of the state and severity of ischemic brain injury.

OUTLOOKS
Temperature is one of the important reference indexes to evaluate healthy states.Whether it is the core body temperature of an individual or the temperature of cellular and subcellular levels, it has been extensively investigated.In recent years, with the development of luminescent nanomaterials, a large number of new nanothermometers have emerged in the field of cell thermometry, indicating a huge demand for cell temperature measurement in basic cell biology research and clinical research.The related nanoparticle thermometers mentioned in this paper are all implemented using fluorescence properties, including fluorescence intensity, fluorescence wavelength shift, ratio fluorescence, fluorescence lifetime, etc., to image cells and measure temperature.In addition, nano-thermometers often need surface modification to make them better for in vivo temperature measurement.In order to specifically target a certain cell or organelle, it usually needs special treatment to meet the needs of the test.
Although these technologies have basically realized cell temperature measurement, the nanothermometers still have some drawbacks.Firstly, the temperature variations are always too small between healthy and diseased states.Presently available nanothermometers are difficult to discriminate these small intracellular temperature changes through optical probes.Secondly, exploration of the relationship between intracellular temperature, cell metabolism, intracellular energy production, transformation and transmission is only superficial and has not been systematically investigated.As for the content of temperature assisted disease treatment, only the application of temperature assisted thermotherapy in the process of tumor hyperthermia has been studied, and there is almost no application in other disease therapy.However, there are a lot of diseases whose treatment and development are closely associated with body or cellular temperature, such as the therapeutic hypothermia of brain injuries and neurological disorders, and the early diagnosis of infectious diseases. 9,130,131Therefore, the use of nanothermometers to reveal physiological state of cells or monitor cellular bioprocess dynamics is an important research trend for nanothermometer-assisted disease therapy.It will pave a way for the realization of an early diagnosis of diseases, and highly precise personalized treatments.
F I G U R E 9 (A) The schematic of in vivo near-infrared (NIR) luminescence imaging for thermometry.(B) NIR-II in vivo bioimaging at 3, 4, 6, 10, and 24 h after reperfusion for the transient middle cerebral artery occlusion (tMCAO) mouse, which was injected with NYCN@PEG at 2 h after reperfusion.(C) Fluorescence microscopy imaging at 3 h after reperfusion for brain tissue section from tMCAO mouse model.(D) NIR luminescencebased temperature imaging of tMCAO mouse injected with NYCN@PEG 3 and 24 h after reperfusion.Reproduced with permission. 129opyright 2023, John Wiley and Sons.

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I G U R E 3 (A) Schematic design of core-shell NaGdF 4 : Yb, Er@NaGdF 4 upconversion luminescent nanoparticles (UCNPs) and energy-transfer process, and the thermally coupled energy levels of 2 H 11/2 and 4 S 3/2 .(B) Cell membrane labeling processes.(C) In situ temperature (T) mapping on the immune cell membrane.Reproduced with permission. 38Copyright 2022, American Chemical Society.(D) Scheme of the temperature measurement of a single living cell with UCNP-decorated spider silk.(E) Optical microscope images of detecting the local temperatures at the membrane near the cell nucleus (R1) and at an extracellular location (R2).(F) Scan of fluorescence intensity ratio (FIR) values around the cell obtained from the spectra at different points along UCNP-decorated spider silk on the cell.

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I G U R E 4 (A) Typical fluorescence lifetime imaging microscopy images of HeLa cells with internalized AuNCs at four different temperatures.Reproduced with permission. 72Copyright 2013, John Wiley and Sons.(B) Schematic image showing the synthetic process and mitochondrial location process of the mAuNCs.(C) Fluorescence lifetime image of the L929 cell incubated with mAuNCs.Reproduced with permission.

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I G U R E 5 (A) Schematic diagram of the synthesis and temperature-responsive fluorescence property of red-emitting carbon nanodots (RCDs) in a solution and within a cell.Reproduced with permission. 82Copyright 2020, American Chemical Society.(B) Temperaturedependent imaging of T-ca cells under excitation of 406 nm, 488 nm, and 561 nm at temperatures of 25˚C, 35˚C, and 45˚C.All scale bars are 100 mm.Reproduced with permission. 83Copyright 2022, Elsevier.All rights reserved.(C) Scheme of the intracellular aggregation of fluorescent nanodiamonds (FNDs).(D) Fluorescence image and temperature mapping of FND aggregates at different locations in a living C127 cell.Reproduced with permission. 84Copyright 2021, Wiley-VCH GmbH.
LIU ET AL.F I G U R E 6 (A) Chemical structures and assembled nanohydrogels of three temperature-sensitive fluorescent polymers (rhodamine Bbased N-isopropylacrylamide polymer (RNP), fluorescein-based N-isopropylacrylamide polymer (FNP), and cage-based Nisopropylacrylamide polymer (CNP)).(B) Fluorescent lifetimes of CNP (blue) at 25 ˚C and nanohydrogels with white-light emission (NWLE) (black) at different temperatures of 25, 32, 37, 42, and 45 ˚C.(C) Commission internationale de l'éclairage (CIE) coordinate diagram of NWLE under 365 nm UV-light irradiation for one heating-cooling cycle.Reproduced with permission. 95Copyright 2022, American Chemical Society.(D) Schematic of the fabrication of the TRF NPs.(E) Photoluminescence spectra of the TRF NPs with the temperature increased from 25 to 65 ˚C.(F) PL intensity ratio versus temperature curves of TRF NPs.Reproduced with permission. 96Copyright 2020, American Chemical Society.T A B L E 1 Different nanothermometers employed for cell thermal sensing.

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I G U R E 8 (A) The time evolution of thermal relaxation normalized difference between tumor tissue and healthy tissue (Δτ, top), the change of tumor volume with time (middle) and the time evolution of the surface temperature difference (bottom).Reproduced with permission. 120Copyright 2018, John Wiley and Sons.(B) Scheme of Photothermal therapy (PTT) tumor treatment with real-time temperature feedback.(C) Temperature increments for different irradiation power densities measured at the tumor site and at the skin surface.(D) Top: evolution of a total control tumor.Bottom: after the Quantum dots (QDs) þ laser (1.7 W cm −2 ) treatment.(E) Evolution of tumors treated with different conditions.Reproduced with permission. 121Copyright 2016, John Wiley and Sons.LIU ET AL.