  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.
This debate has led to a hypothesis that intracellular thermal conduction is
considerably slower than in water. Although various efforts have been made to
measure intracellular thermal conduction, there are significant variations in
the reported values. 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. We
measured intracellular thermal diffusivity via transient thermal decay and
found that thermal diffusivity within living cells is 93-94% of water in the
cytoplasm and nucleus. This result suggests that intracellular thermal
conduction cannot explain the 10^5 gap issue. To investigate this issue
further, we compared fluorescent nanothermometry with our label-free
thermometry by measuring heat-induced temperature variations in living cells.
This experiment revealed intriguing results where the fluorescent
nanothermometry showed a slowly varying unexpected signal in addition to a
constant temperature change. This result indicates that fluorescent
nanothermometers are sensitive to additional factors on top of the temperature
variation under local thermal equilibrium, raising an open question of what
fluorescent nanothermometry measures.
