Nanothermometry in Living Cells: Physical Limits, Conceptual and Material Challenges

ABSTRACT Reports of large temperature inhomogeneities inside single cells have sparked intense debate over the past decade. Initial claims were challenged as inconsistent with basic heat transport, yet the number of such reports continues to grow. Here, we present a concise, physics‐led assessment of intracellular nanothermometry, grounded in experimental data from cellular biology and studies reporting intracellular temperature contrast, thermal diffusivity, and thermal conductivity. This perspective provides a stricter and more detailed treatment of physical models, including a microscopic approach to thermal conductivity and molecular dynamics simulations. We also review potential sources of biased measurements in cells, including photonic effects, osmotic changes, optical trapping by tightly focused beams (tweezer effect), and electric‐field shifts. Diamond color centers are highlighted as promising candidates for reference thermometers. Nitrogen‐vacancy (NV) centers enable optical readout through microwave‐driven resonance that is largely insensitive to optical inhomogeneities and allows in situ cross‐checks using multiple centers (NV, SiV, and others) embedded in the same nanocrystal. Their sensitivity to electric fields can be reduced by surface passivation, improving the reliability of intracellular temperature measurements. However, challenges in materials science, fabrication, and, most importantly, quantitative theoretical modeling remain to be addressed.