Daniel Jaque

Intracellular luminescence thermometry has long promised to reveal how heat is generated, dissipated, and regulated inside living cells. Yet, despite substantial progress, the field remains shaped by disagreement over the magnitude and physical plausibility of reported intracellular temperature gradients. In this manuscript, we discuss luminescence thermometry as a powerful approach for probing temperature at subcellular length scales, while emphasizing the experimental care required to make such measurements meaningful. After outlining the field’s evolution, we summarize the relevant heat transfer concepts, before introducing luminescence thermometry and defining the performance metrics used to describe readout uncertainty and accuracy within a metrological framework. We then examine how thermometer design, intracellular localization, calibration, microscopy configuration, and data treatment influence the final thermal readout. Particular attention is given to sources of loss in readout accuracy. Among them, we highlight cross-sensitivity, which arises when the probe responds to parameters other than temperature, such as pH, viscosity, ionic strength, or biomolecular interactions. Finally, we propose practical directions for improving reproducibility, including multifeature readouts, machine-learning-assisted analysis, and FAIR data practices, while suggesting future research directions.

Near‐infrared (NIR)‐emitting Ag ₂ S nanocrystals (NCs) are promising contrast agents in nanomedicine, thanks to extensive efforts to improve their photochemical stability, biocompatibility, and brightness by tuning their size, composition, surface chemistry, and core/shell architecture. However, when dispersed in biological media, these NCs undergo non‐specific interactions with surrounding species, including biomolecules. Such interactions may compromise their colloidal and physicochemical stability, alter their photoluminescence properties, and impair both in vitro and in vivo imaging and sensing performance. Here, we show that incorporating NIR‐emitting Ag ₂ S‐based NCs into colloidal dendritic silica effectively minimizes environmental interference in complex biological media, including blood plasma and whole blood. Steady‐state and time‐resolved photoluminescence measurements reveal that both emission intensity and lifetime are substantially better preserved following incorporation into silica colloids. This preservation has a dual benefit: maintaining photoluminescence intensity enables precise NIR imaging, while retention of spectral and temporal responses preserves the NCs’ thermal sensing capability. Proof‐of‐concept experiments in tissue phantoms and small‐animal models confirm these findings. The approach supports reliable optical readouts under biologically relevant conditions without modifying the NC composition. Overall, incorporation into dendritic silica colloids offers a viable strategy for protecting luminescent nanoparticles from biological interference and retaining their optical properties for biomedical imaging and sensing applications.

Plasmonic trapping enables nanoscale confinement of individual nanoparticles by exploiting localized electromagnetic field enhancement, an effect inherently accompanied by photothermal heating. Quantifying this local temperature increase is essential, as heating can affect trap stability through enhanced Brownian motion and the emergence of thermally induced forces such as thermophoresis. Here, we experimentally determine the local temperature experienced by a single upconverting nanoparticle optically trapped in the vicinity of a pyramid-like gold nanoantenna. By combining this measurement with numerical simulations of the heat distribution, we infer the temperature at the gold–water interface. While the trapped UCNP experiences a temperature increase of ∼40 °C at an average distance of ∼50 nm from the nanoantenna surface, thermal simulations predict that the nanoantenna surface may reach temperatures of up to ∼100 °C under the same excitation condition. Analysis of the nanoparticle’s luminescence provides simultaneous determination of its local temperature and three-dimensional motion in the vicinity of the nanoantenna. The results reveal a linear increase in temperature with trapping power and demonstrate that the particle explores regions characterized by strong thermal gradients generated by the plasmonic structure. By correlating temperature and position, we estimate the relative magnitudes of optical and thermophoretic forces, providing a model-based explanation for the observed steady-state trapping position. These findings establish individual upconverting nanoparticles as probes for investigating thermoplasmonic effects at the single-particle level and provide insight into the interplay between optical confinement, thermal gradients, and Brownian motion in plasmonic trapping systems.

ConspectusLanthanide-doped upconverting nanoparticles (UCNPs) convert near-infrared (NIR) light into visible emission through multiphoton absorption processes. This property has made them useful tools for sensing and single-particle studies in biological environments. Their characteristic narrow emission lines, resistance to photobleaching, and the thermal coupling between the excited states of trivalent erbium (Er³⁺) have positioned UCNPs as a practical platform for intracellular thermometry despite their moderate sensitivity. Their response can be measured with standard microscopes using inexpensive NIR excitation sources, enabling experiments that are not easily achieved with other types of nanoscale thermometers. However, using UCNPs inside living cells also reveals important limitations because the intracellular medium is chemically complex and may alter the emission properties that form the basis of the thermal readout. Variations in local pH, ion concentration, viscosity, or molecular crowding can shift energy levels or modify nonradiative relaxation pathways. As a result, establishing the reliability of UCNP thermometry inside cells requires dedicated studies on particle stability, surface chemistry, and the influence of the cytoplasmic environment. Understanding these effects is essential for determining whether ratiometric UCNP thermometry can serve as a quantitative intracellular tool rather than only a qualitative indicator of heating. UCNPs have also enabled new single-particle experiments through optical trapping. A tightly focused NIR beam can immobilize an individual nanoparticle while simultaneously exciting its upconversion emission, allowing continuous monitoring of spectral changes, rotational dynamics, and local mechanical properties. These studies provide access to information that is hidden in ensemble measurements, such as particle-particle radiative interactions or the coupling between particle rotation/movement and local viscosity. Yet optical trapping also brings its own challenges: the forces acting on sub-100 nm UCNPs are modest, and trapping stability is strongly affected by laser-induced heating. Increasing dopant concentration improves confinement but raises thermal load, leading to a trade-off that must be carefully balanced. Approaches based on plasmonic enhancement are not suitable due to their additional heating, motivating interest in alternative photonic structures such as dielectric metasurfaces that could strengthen confinement while keeping temperatures low. Together, intracellular thermometry and single-particle trapping illustrate both the strengths and the current limitations of UCNPs. Their optical properties make them accessible probes for measuring local temperature and mechanical responses, but their performance heavily depends on the surrounding environment. In this Account, we summarize our efforts to understand UCNP behavior under realistic biological and trapping conditions. We discuss the stability of the thermometric response inside cells, the confinement-heating balance in optical traps, and the opportunities offered by single-particle measurements for studying light-matter interactions at the nanoscale. These considerations outline a path toward more reliable sensing and manipulation strategies based on UCNPs.

Near-infrared (NIR) imaging and related sensing technologies must operate under biologically constrained conditions of low excitation power and strong tissue scattering, making efficient light extraction crucial. Here, we show that a simple architecture of Ag2S-based nanoparticles deposited on metallic reflective surfaces markedly amplifies the photoluminescence (PL) signal through a mirror-induced optical feedback effect. This passive amplification enhances the signal-to-noise ratio (SNR) and allows detection through cm-thick tissue-mimicking media under low excitation power and short integration times. Furthermore, the increased SNR enables the temperature-dependent PL contrast to be quantified more reliably over the working temperature range, without modifying the intrinsic photophysical response of the emitters. Integrating nanothermometers with metals as passive optical amplifiers thus provides a scalable route toward high-performance, bio-integrated photonic platforms for NIR imaging and thermometry, under realistic, tissue-relevant conditions.

Optical tweezers use focused laser beams to manipulate small particles, primarily for force sensing. Recent advances in nanoscale-trapping approaches have enabled the development of multiplexed sensing applications, such as temperature and viscosity detection. Upconversion particles (UCPs) and, in particular, lanthanide-doped nano-/micro-crystals (~6 nm to 6 μm) exhibit particular anti-Stokes emission properties, which facilitate their visualization when trapped and the detection of changes to their properties based on temperature and orientation. Their ion resonance enhances the trapping force, enabling the manipulation of smaller particles and their use for force sensing. Here we provide step-by-step instructions to build UCP-based holographic optical tweezers systems, including super-resolved photonic force microscopy and fluorescence optical tweezers. We detail the characterization of the setup for subfemtonewton-scale force sensing and include nanoprobe functionalization, force sensitivity validation and comparison with known forces. We further include the procedures for temperature and viscosity sensing, such as calibrating polarized spectra, initiating UCP rotation and analyzing viscosity via spectral fluctuations. Applications, including nanoparticle-DNA-coated gold film interactions and temperature distribution near single cells, are shown as well. The procedure typically requires 6 days to complete and is suitable for users with expertise in photonics.

Bioorthogonal click chemistry enables selective detection of VCAM-1 overexpression in endothelial cells using luminescent nanothermometers.

Accurate, rapid, and remote temperature sensing at the nanoscale is essential for applications ranging from monitoring cellular thermodynamics to thermal management of microelectronic devices. Luminescent nanothermometers are promising candidates; however, their deployment is hindered by limited thermal sensitivity and cross-sensitivity to environmental factors that mimic temperature-induced luminescence changes. We introduce fluorescent chromatic nanoswitchers (CNSs), comprising silica nanocapsules incorporating a fluorescent dye within a thermoresponsive matrix. The matrix undergoes a solid-to-liquid phase transition, yielding an exceptional fluorescence lifetime sensitivity of 19% °C^-1 at 37 °C. Crucially, the lifetime-based thermal readout provided by CNSs is resistant to environmental interference, ensuring reliable, reproducible temperature measurements. To enhance CNS performance, we integrate artificial neural networks (ANNs) for advanced lifetime signal processing, enabling faster and robust thermal readouts. Proof-of-concept experiments show that the synergy between high-sensitivity lifetime-based nanothermometers and ANN-driven analysis paves the way for next-generation thermal sensing technologies, offering improved responsiveness, real-time capabilities, and enhanced accuracy.

In modern environments, the brain is continuously exposed to numerous external stimuli, including the microwave radiation used in telecommunication technologies. It has been suggested that the absorption of this radiation by brain tissue can induce local heating. Because brain temperature influences neural activity, metabolism, and overall brain function, microwave‐induced heating raises concerns over the safety of such technologies. Proper evaluation of the risks associated with microwave‐based technologies thus requires accurate quantification of heating in deep organs without disrupting their physiology. This study, demonstrates that microwave‐induced brain heating can be remotely monitored in vivo via luminescence thermometry using near‐infrared luminescent silver sulfide (Ag ₂ S) nanoparticles. Their temperature‐dependent luminescence lifetime is a reliable thermometric parameter for the measurement of absolute brain temperature. The in vivo results offer direct, real‐time evidence of brain heating (up to 4 °C) under telecom exposure conditions (3 GHz). Moreover, they establish lifetime thermometry as a reliable, minimally invasive approach for investigating thermoregulation in deep tissues even under external electromagnetic stimulation.