Intracellular luminescence thermometry: A story of disagreement, trust, and hope
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 development, we outline the relevant heat-transfer concepts, before introducing luminescence thermometry and the performance metrics used to describe precision and accuracy. We then examine how thermometer design, intracellular localization, calibration, microscopy configuration, and data treatment influence the final thermal readout. Particular attention is given to two recurrent sources of error: bias, arising from measurement conditions and optical distortions, and cross-sensitivity, arising when the probe responds to parameters other than temperature, such as pH, viscosity, ionic strength, or biomolecular interactions. Finally, we outline practical directions for improving reproducibility, including multi-feature readouts, machine-learning-assisted analysis, and FAIR data practices, while suggesting future research directions.