Label-free mid-infrared photothermal microscopy revisits intracellular thermal dynamics: what do fluorescent nanothermometers measure? (2406.16265v2)

Abstract: Fluorescent nanothermometry has revealed pronounced inhomogeneous temperature distributions within cells and has opened the field of single-cell thermal biology. However, this finding has sparked a controversial discussion known as the 10^5 gap issue, which arises from a simple heat conduction calculation suggesting such large temperature distributions should not exist inside cells. Here, we address this issue using label-free mid-infrared photothermal microscopy, which enables us to measure heat-induced temperature variations under local thermal equilibrium via refractive index changes. First, we measured intracellular thermal diffusivity via transient thermal decay and determined that thermal diffusivity within living cells is 93-94% of water in the cytoplasm and nucleus. This result does not support the hypothesis that intracellular thermal conduction is considerably slower than in water. Next, we compared fluorescent nanothermometry with our label-free thermometry by measuring heat-induced temperature variations in living cells and found that the fluorescent nanothermometry showed a slowly varying signal in addition to a rapidly responding temperature change. This result indicates that fluorescent nanothermometers are sensitive to additional factors beyond the temperature defined under local thermal equilibrium, which do not follow the behavior predicted by thermal conduction. From this study, we conclude that the 10^5 gap issue arises from comparing two fundamentally different physical quantities. This finding also raises an open question regarding the origin of the additional factors that fluorescence nanothermometers measure.