Intracellular Luminescence Thermometry: A Story of Disagreement, Trust, and Hope

Abstract 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.