Label‐Free Imaging of Intracellular Temperature by Using the O−H Stretching Raman Band of Water

We propose a label‐free method for measuring intracellular temperature using a Raman image of a cell in the O?H stretching band. Raman spectra of cultured cells and the medium were first measured at various temperatures using a Raman microscope and the intensity ratio of the two regions of the O?H stretching band was calculated. The intensity ratio varies linearly with temperature in both the medium and cells, and the resulting calibration lines allow simultaneous visualization of both intracellular and extracellular temperatures in a label‐free manner. We applied this method to the measurement of temperature changes after the introduction of FCCP (carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone) in living cells. We observed a temperature rise in the cytoplasm and succeeded in obtaining an image of the change in intracellular temperature after the FCCP treatment.