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Intracellular temperature is a fundamental parameter in biochemical reactions. Genetically encoded fluorescent temperature indicators (GETIs) have been developed to visualize intracellular thermogenesis; however, the temperature sensitivity or localization capability in specific organelles should have been further improved to clearly capture when and where intracellular temperature changes at the subcellular level occur. Here, we developed a new GETI, gMELT, composed of donor and acceptor subunits, in which cyan and yellow fluorescent proteins, respectively, as a Förster resonance energy transfer (FRET) pair were fused with temperature-sensitive domains. The donor and acceptor subunits associated and dissociated in response to temperature changes, altering the FRET efficiency. Consequently, gMELT functioned as a fluorescence ratiometric indicator. Untagged gMELT was expressed in the cytoplasm, whereas versions fused with specific localization signals were targeted to the endoplasmic reticulum (ER) or mitochondria. All gMELT variations enabled more sensitive temperature measurements in cellular compartments than those in previous GETIs. The gMELTs, tagged with ER or mitochondrial targeting sequences, were used to detect thermogenesis in organelles stimulated chemically, a method previously known to induce thermogenesis. The observed temperature changes were comparable to previous reports, assuming that the fluorescence readout changes were exclusively due to temperature variations. Furthermore, we demonstrated how macromolecular crowding influences gMELT fluorescence given that this factor can subtly affect the fluorescence readout. Investigating thermogenesis with gMELT, accounting for factors such as macromolecular crowding, will enhance our understanding of intracellular thermogenesis phenomena.

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Despite significant advances, cancer remains a leading global cause of death. Current therapies often fail due to incomplete tumor removal and nonspecific targeting, spurring interest in alternative treatments. Hyperthermia, which uses elevated temperatures to kill cancer cells or boost their sensitivity to radio/chemotherapy, has emerged as a promising alternative. Recent advancements employ nanoparticles (NPs) as heat mediators for selective cancer cell destruction, minimizing damage to healthy tissues. This approach, known as NP hyperthermia, falls into two categories: photothermal therapies (PTT) and magnetothermal therapies (MTT). PTT utilizes NPs that convert light to heat, while MTT uses magnetic NPs activated by alternating magnetic fields (AMF), both achieving localized tumor damage. These methods offer advantages like precise targeting, minimal invasiveness, and reduced systemic toxicity. However, the efficacy of NP hyperthermia depends on many factors, in particular, the NP properties, the tumor microenvironment (TME), and TME-NP interactions. Optimizing this treatment requires accurate heat monitoring strategies, such as nanothermometry and biologically relevant screening models that can better mimic the physiological features of the tumor in the human body. This review explores the state-of-the-art in NP-mediated cancer hyperthermia, discussing available nanomaterials, their strengths and weaknesses, characterization methods, and future directions. Our particular focus lies in preclinical NP screening techniques, providing an updated perspective on their efficacy and relevance in the journey towards clinical trials.

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Temperature regulates nonradiative processes in luminescent materials, fundamental to luminescence nanothermometry. However, elevated temperatures often suppress the radiative process, limiting the sensitivity of thermometers. Here, we introduce an approach to populating the excited state of lanthanides at elevated temperatures, resulting in a sizable lifetime lengthening and intensity increase of the near-infrared (NIR)-II emission. The key is to create a five-energy-level system and use a pair of lanthanides to leverage the cross-relaxation process. We observed the lifetime of NIR-II emission of Er3+ has been remarkably increased from 3.85 to 7.54 ms by codoping only 0.5 mol % Ce3+ at 20 °C and further increased to 7.80 ms when increasing the temperature to 40 °C. Moreover, this concept is universal across four ion pairs and remains stable within aqueous nanoparticles. Our findings emphasize the need to design energy transfer systems that overcome the constraint of thermal quenching, enabling efficient imaging and sensing.

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The development of efficient nanoscale photon absorbers, such as plasmonic or high-index dielectric nanostructures, allows the remotely controlled release of heat on the nanoscale using light. These photothermal nanomaterials have found applications in various research and technological fields, ranging from materials science to biology. However, measuring the nanoscale thermal fields remains an open challenge, hindering full comprehension and control of nanoscale photothermal phenomena. Here, we review and discuss existent thermometries suitable for single nanoparticles heated under illumination. These methods are classified in four categories according to the region where they assess temperature: (1) the average temperature within a diffraction-limited volume, (2) the average temperature at the immediate vicinity of the nanoparticle surface, (3) the temperature of the nanoparticle itself, and (4) a map of the temperature around the nanoparticle with nanoscale spatial resolution. In the latter, because it is the most challenging and informative type of method, we also envisage new combinations of technologies that could be helpful in retrieving nanoscale temperature maps. Finally, we analyze and provide examples of strategies to validate the results obtained using different thermometry methods.

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The short-wave infrared (SWIR) photoluminescence lifetimes of rare-earth doped nanoparticles (RENPs) have found diverse applications in fundamental and applied research. Despite dazzling progress in the novel design and synthesis of RENPs with attractive optical properties, existing optical systems for SWIR photoluminescence lifetime imaging are still considerably restricted by inefficient photon detection, limited imaging speed, and low sensitivity. To overcome these challenges, SWIR photoluminescence lifetime imaging microscopy using an all-optical streak camera (PLIMASC) is developed. Synergizing scanning optics and a high-sensitivity InGaAs CMOS camera, SWIR-PLIMASC has a 1D imaging speed of up to 138.9 kHz in the spectral range of 900-1700 nm, which quantifies the photoluminescence lifetime of RENPs in a single shot. A 2D photoluminescence lifetime map can be acquired by 1D scanning of the sample. To showcase the power of SWIR-PLIMASC, a series of core-shell RENPs with distinct SWIR photoluminescence lifetimes is synthesized. In particular, using Er3+ -doped RENPs, SWIR-PLIMASC enables multiplexed anti-counterfeiting. Leveraging Ho3+ -doped RENPs as temperature indicators, this system is applied to SWIR photoluminescence lifetime-based thermometry. Opening up a new avenue for efficient SWIR photoluminescence lifetime mapping, this work is envisaged to contribute to advanced materials characterization, information science, and biomedicine.

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When navigated by the available energy of a system, often provided in the form of heat, physical processes or chemical reactions fleet on a free-energy landscape, thus changing the structure. In in situ transmission electron microscopy (TEM), where material structures are measured and manipulated inside the microscope while being subjected to external stimuli such as electrical fields, laser irradiation, or mechanical stress, it is necessary to precisely determine the local temperature of the specimen to provide a comprehensive understanding of material behavior and to establish the relationship among energy, structure, and properties at the nanoscale. Here, we propose using cathodoluminescence (CL) spectroscopy in TEM for in situ measurement of the local temperature. Gadolinium oxide particles doped with emissive europium ions present an opportunity to utilize them as a temperature probe in CL measurements via a ratiometric approach. We show the thermometric performance of the probe and demonstrate a precision of ±5 K in the temperature range from 113 to 323 K with the spatial resolution limited by the size of the particles, which surpasses other methods for temperature determination. With the CL-based thermometry, we further demonstrate measuring local temperature under laser irradiation.

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Luminescent nanothermometry, particularly the one based on ratiometric, has sparked intense research for non-invasive in vivo or intracellular temperature mapping, empowering their uses as diagnosis tools in biomedicine. However, ratiometric detection still suffers from biased sensing induced by wavelength-dependent tissue absorption and scattering, low thermal sensitivity (Sr ), and lack of imaging depth information. Herein, this work constructs an ultrasensitive NIR-II ratiometric nanothermometer with self-calibrating ability for 3D in vivo thermographic imaging, in which temperature-insensitive lanthanide nanocrystals and strongly temperature-quenched Ag2 S quantum dots are co-assembled to form a hybrid nanocomposite material. Precise control over the amount ratio between two sub-materials enables the manipulation of heat-activated back energy transfer from Ag2 S to Yb3+ in lanthanide nanoparticles, thereby rendering Sr up to 7.8% °C-1 at 43.5 °C, and higher than 6.5% °C-1 over the entire physiological temperature range. Moreover, the luminescence intensity ratio between two separated spectral regions within the narrow Yb3+ emission peak is used to determine the depth information of nanothermometers in living mice and correct the effect of tissue depth on 2D thermographic imaging, and therefore allows a proof-of-concept demonstration of accurate 3D in vivo thermographic imaging, constituting a solid step toward the development of advanced ratiometric nanothermometry for biological applications.

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Anisotropic hybrid nanostructures stand out as promising therapeutic agents in photothermal conversion-based treatments. Accordingly, understanding local heat generation mediated by light-to-heat conversion of absorbing multicomponent nanoparticles at the single-particle level has forthwith become a subject of broad and current interest. Nonetheless, evaluating reliable temperature profiles around a single trapped nanoparticle is challenging from all of the experimental, computational, and fundamental viewpoints. Committed to filling this gap, the heat generation of an anisotropic hybrid nanostructure is explored by means of two different experimental approaches from which the local temperature is measured in a direct or indirect way, all in the context of hot Brownian motion theory. The results were compared with analytical results supported by the numerical computation of the wavelength-dependent absorption efficiencies in the discrete dipole approximation for scattering calculations, which has been extended to inhomogeneous nanostructures. Overall, we provide a consistent and comprehensive view of the heat generation in optical traps of highly absorbing particles from the viewpoint of the hot Brownian motion theory.

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Temperature is a crucial regulator of the rate and direction of biochemical reactions and cell processes. The recent data indicating the presence of local thermal gradients associated with the sites of high-rate thermogenesis, on the one hand, demonstrate the possibility for the existence of "thermal signaling" in a cell and, on the other, are criticized on the basis of thermodynamic calculations and models. Here, we review the main thermometric techniques and sensors developed for the determination of temperature inside living cells and diverse intracellular compartments. A comparative analysis is conducted of the results obtained using these methods for the cytosol, nucleus, endo-/sarcoplasmic reticulum, and mitochondria, as well as their biological consistency. Special attention is given to the limitations, possible sources of errors and ambiguities of the sensor's responses. The issue of biological temperature limits in cells and organelles is considered. It is concluded that the elaboration of experimental protocols for ultralocal temperature measurements that take into account both the characteristics of biological systems, as well as the properties and limitations of each type of sensor is of critical importance for the generation of reliable results and further progress in this field.

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Luminescence (nano)thermometry is a remote sensing technique that relies on the temperature dependency of the luminescence features (e.g., bandshape, peak energy or intensity, and excited state lifetimes and risetimes) of a phosphor to measure temperature. This technique provides precise thermal readouts with superior spatial resolution in short acquisition times. Although luminescence thermometry is just starting to become a more mature subject, it exhibits enormous potential in several areas, e.g., optoelectronics, photonics, micro- and nanofluidics, and nanomedicine. This work reviews the latest trends in the field, including the establishment of a comprehensive theoretical background and standardized practices. The reliability, repeatability, and reproducibility of the technique are also discussed, along with the use of multiparametric analysis and artificial-intelligence algorithms to enhance thermal readouts. In addition, examples are provided to underscore the challenges that luminescence thermometry faces, alongside the need for a continuous search and design of new materials, experimental techniques, and analysis procedures to improve the competitiveness, accessibility, and popularity of the technology.

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Temperature plays a critical role in regulating body mechanisms and indicating inflammatory processes. Local temperature increments above 42 °C are shown to kill cancer cells in tumorous tissue, leading to the development of nanoparticle-mediated thermo-therapeutic strategies for fighting oncological diseases. Remarkably, these therapeutic effects can occur without macroscopic temperature rise, suggesting localized nanoparticle heating, and minimizing side effects on healthy tissues. Nanothermometry has received considerable attention as a means of developing nanothermosensing approaches to monitor the temperature at the core of nanoparticle atoms inside cells. In this study, a label-free, direct, and universal nanoscale thermometry is proposed to monitor the thermal processes of nanoparticles under photoexcitation in the tumor environment. Gold-iron oxide nanohybrids are utilized as multifunctional photothermal agents internalized in a 3D tumor model of glioblastoma that mimics the in vivo scenario. The local temperature under near-infrared photo-excitation is monitored by X-ray absorption spectroscopy (XAS) at the Au L3 -edge (11 919 eV) to obtain their temperature in cells, deepening the knowledge of nanothermal tumor treatments. This nanothermometric approach demonstrates its potential in detecting high nanothermal changes in tumor-mimicking tissues. It offers a notable advantage by enabling thermal sensing of any element, effectively transforming any material into a nanothermometer within biological environments.

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The fields of biosensitivity and biological imaging have received a lot of attention from rare earth-doped upconversion nanoparticles (UCNPs). However, owing to the relatively large energy difference of rare earth ions, biological sensitivity based on UCNPs is restricted to detect at low temperature. Here, we design core-shell-shell NaErF4:Yb@Nd2O3@SiO2 UCNPs as a dual-mode bioprobe that produces blue, green, and red multi-color upconversion emissions at extremely low temperatures between 100 K and 280 K. Based on the thermally coupled energy levels (TCELs) of Er3+ (2H11/2 and 4S3/2) and Nd3+ (4F5/2 and 4F3/2) at 100 K, the greatest relative sensitivity (SR) approaches 12.7% K-1. NaErF4:Yb@Nd2O3@SiO2 injection is used to achieve blue upconversion emission imaging of frozen heart tissue, showing that this UCNP can serve as a low-temperature sensitive biological fluorescence.

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In nanothermometry, the use of nanoparticles as thermal probes enables remote and minimally invasive sensing. In the biomedical context, nanothermometry has emerged as a powerful tool where traditional approaches, like infrared thermal sensing and contact thermometers, fall short. Despite the strides of this technology in preclinical settings, nanothermometry is not mature enough to be translated to the bedside. This is due to two major hurdles: the inability to perform 3D thermal imaging and the requirement for tools that are readily available in the clinics. This work simultaneously overcomes both limitations by proposing the technology of optical coherence thermometry (OCTh). This is achieved by combining thermoresponsive polymeric nanogels and optical coherence tomography (OCT)-a 3D imaging technology routinely used in clinical practice. The volume phase transition of the thermoresponsive nanogels causes marked changes in their refractive index, making them temperature-sensitive OCT contrast agents. The ability of OCTh to provide 3D thermal images is demonstrated in tissue phantoms subjected to photothermal processes, and its reliability is corroborated by comparing experimental results with numerical simulations. The results included in this work set credible foundations for the implementation of nanothermometry in the form of OCTh in clinical practice.

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In the last two decades, advances in the dark field detectors and microscopes of scanning transmission electron microscopy (STEM) have inspired a resurgence of interest in quantitative STEM analysis. One promising avenue is the use of STEM as a nanothermometric probe. In this application, thermal diffuse scattering, captured by a CCD camera or an annular dark field detector, acts as an indirect measurement of the specimen temperature. One challenge with taking such a measurement is achieving adequate sensitivity to quantify a change in scattered electron signal on the order of 1% or less of the full electron beam. Another difficulty is decoupling the thermal effect on electron scattering from scattering changes due to differing specimen thicknesses and materials. To address these issues, we have developed a method using STEM, combined with electron energy loss spectroscopy (EELS), to produce a material-specific calibration curve. On silicon, across the range 89 K to 294 K, we measured a monotonically increasing HAADF signal ranging from 4.0% to 4.4% of the direct beam intensity at a thickness-to-mean-free-path ratio of 0.5. This yielded a calibration curve of temperature versus full-beam-normalized, thickness-normalized HAADF signal. The method enables thermal measurements on a specimen of varying local thickness at a spatial resolution of a few nanometers. We demonstrated the potential of the technique for testing electron scattering models by applying single-electron scattering theory to the data collected to extract a measurement of the mean atomic vibration amplitude in silicon at 294 K. The measured value, 0.00738 ± 0.00002 nm, agrees well with reported measurement using X-rays.

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The intracellular metabolism of organelles, like lysosomes and mitochondria, is highly coordinated spatiotemporally and functionally. The activities of lysosomal enzymes significantly rely on the cytoplasmic temperature, and heat is constantly released by mitochondria as the byproduct of adenosine triphosphate (ATP) generation during active metabolism. Here, we developed temperature-sensitive LysoDots and MitoDots to monitor the in situ thermal dynamics of lysosomes and mitochondria. The design is based on upconversion nanoparticles (UCNPs) with high-density surface modifications to achieve the exceptionally high sensitivity of 2.7% K-1 and low uncertainty of 0.8 K for nanothermometry to be used in living cells. We show the measurement is independent of the ion concentrations and pH values. With Ca2+ ion shock, the temperatures of both lysosomes and mitochondria increased by ∼2 to 4 °C. Intriguingly, with chloroquine (CQ) treatment, the lysosomal temperature was observed to decrease by up to ∼3 °C, while mitochondria remained relatively stable. Lastly, with oxidative phosphorylation inhibitor treatment, we observed an ∼3 to 7 °C temperature increase and a thermal transition from mitochondria to lysosomes. These observations indicate different metabolic pathways and thermal transitions between lysosomes and mitochondria inside HeLa cells. The nanothermometry probes provide a powerful tool for multimodality functional imaging of subcellular organelles and interactions with high spatial, temporal, and thermal dynamics resolutions.

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Multifunctional nano-objects containing a magnetic heater and a temperature emissive sensor in the same nanoparticle have recently emerged as promising tools towards personalized nanomedicine permitting hyperthermia-assisted treatment under local temperature control. However, a fine control of nano-systems' morphology permitting the synthesis of a single magnetic core with controlled position of the sensor presents a main challenge. We report here the design of new iron oxide core-silica shell nano-objects containing luminescent Tb3+/Eu3+-(acetylacetonate) moieties covalently anchored to the silica surface, which act as a promising heater/thermometer system. They present a single magnetic core and a controlled thickness of the silica shell, permitting a uniform spatial distribution of the emissive nanothermometer relative to the heat source. These nanoparticles exhibit the Tb3+ and Eu3+ characteristic emissions and suitable magnetic properties that make them efficient as a nanoheater with a Ln3+-based emissive self-referencing temperature sensor covalently coupled to it. Heating capacity under an alternating current magnetic field was demonstrated by thermal imaging. This system offers a new strategy permitting a rapid heating of a solution under an applied magnetic field and a local self-referencing temperature sensing with excellent thermal sensitivity (1.64%·K-1 (at 40 °C)) in the range 25-70 °C, good photostability, and reproducibility after several heating cycles.

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Thermal activation of upconversion luminescence in nanocrystals opens up new opportunities in biotechnology and nanophotonics. However, it remains a daunting challenge to achieve a smart control of luminescence behavior in the thermal field with remarkable enhancement and ultrahigh sensitivity. Moreover, the physical picture involved is also debatable. Here we report a novel mechanistic design to realize an ultrasensitive thermally activated upconversion in an erbium sublattice core-shell nanostructure. By enabling a thermosensitive property into the intermediate 4I11/2 level of Er3+ through an energy-migration-mediated surface interaction, the upconverted luminescence was markedly enhanced in the thermal field together with a striking thermochromic feature under 1530 nm irradiation. Importantly, the use of non thermally coupled red and green emissions contributes to the thermal sensitivity up to 5.27% K-1, 3 times higher than that obtained by using conventional thermally coupled green emissions. We further demonstrate that the controllable surface interaction is a general approach to the thermal enhancement of upconversion for a series of lanthanide-based nanomaterials. Our findings pave a new way for the development of smart luminescent materials toward emerging applications such as noncontact nanothermometry, information security, and anticounterfeiting.

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Biofunctionalized nanoparticles are increasingly used in biomedical applications including sensing, targeted delivery, and hyperthermia. However, laser excitation and associated heating of the nanomaterials may alter the structure and interactions of the conjugated biomolecules. Currently no method exists that directly monitors the local temperature near the material's interface where the conjugated biomolecules are. Here, a nanothermometer is reported based on DNA-mediated points accumulation for imaging nanoscale topography (DNA-PAINT) microscopy. The temperature dependent kinetics of repeated and reversible DNA interactions provide a direct readout of the local interfacial temperature. The accuracy and precision of the method is demonstrated by measuring the interfacial temperature of many individual gold nanoparticles in parallel, with a precision of 1 K. In agreement with numerical models, large particle-to-particle differences in the interfacial temperature are found due to underlying differences in optical and thermal properties. In addition, the reversible DNA interactions enable the tracking of interfacial temperature in real-time with intervals of a few minutes. This method does not require prior knowledge of the optical and thermal properties of the sample, and therefore opens the window to understanding and controlling interfacial heating in a wide range of nanomaterials.

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Lanthanide-based upconverting nanoparticles (UCNPs) are trustworthy workhorses in luminescent nanothermometry. The use of UCNPs-based nanothermometers has enabled the determination of the thermal properties of cell membranes and monitoring of in vivo thermal therapies in real time. However, UCNPs boast low thermal sensitivity and brightness, which, along with the difficulty in controlling individual UCNP remotely, make them less than ideal nanothermometers at the single-particle level. In this work, it is shown how these problems can be elegantly solved using a thermoresponsive polymeric coating. Upon decorating the surface of NaYF4 :Er3+ ,Yb3+ UCNPs with poly(N-isopropylacrylamide) (PNIPAM), a >10-fold enhancement in optical forces is observed, allowing stable trapping and manipulation of a single UCNP in the physiological temperature range (20-45 °C). This optical force improvement is accompanied by a significant enhancement of the thermal sensitivity- a maximum value of 8% °C+1 at 32 °C induced by the collapse of PNIPAM. Numerical simulations reveal that the enhancement in thermal sensitivity mainly stems from the high-refractive-index polymeric coating that behaves as a nanolens of high numerical aperture. The results in this work demonstrate how UCNP nanothermometers can be further improved by an adequate surface decoration and open a new avenue toward highly sensitive single-particle nanothermometry.

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The temperature of nanoparticles is a critical parameter in applications that range from biology, to sensors, to photocatalysis. Yet, accurately determining the absolute temperature of nanoparticles is intrinsically difficult because traditional temperature probes likely deliver inaccurate results due to their large thermal mass compared to the nanoparticles. Here we present a hydrogen nanothermometry method that enables a noninvasive and direct measurement of absolute Pd nanoparticle temperature via the temperature dependence of the first-order phase transformation during Pd hydride formation. We apply it to accurately measure light-absorption-induced Pd nanoparticle heating at different irradiated powers with 1 °C resolution and to unravel the impact of nanoparticle density in an array on the obtained temperature. In a wider perspective, this work reports a noninvasive method for accurate temperature measurements at the nanoscale, which we predict will find application in, for example, nano-optics, nanolithography, and plasmon-mediated catalysis to distinguish thermal from electronic effects.

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