A Simple Calibration Method of Anti-Stokes–Stokes Raman Intensity Ratios Using the Water Spectrum for Intracellular Temperature Measurements

Presented here is a facile and practical method for calibrating anti-Stokes–Stokes intensity ratios in low-frequency Raman spectra that is devised specifically for temperature measurements inside cells. The proposed method uses as an intensity standard the low-frequency Raman spectrum of liquid water, a major molecular component of cells, whose temperature is independently measured with a thermocouple. Rather than calibrating pixel intensities themselves, we obtain a correction factor at each Raman shift in the 20–200 cm−1 region by dividing the anti-Stokes–Stokes intensity ratio calculated theoretically from the Boltzmann factor at the known temperature by that obtained experimentally. The validity of the correction curve so obtained is confirmed by measuring water at other temperatures. The anti-Stokes–Stokes intensity ratios that have been subjected to our calibration are well fitted with the Boltzmann factor within ∼1% errors and yield water temperatures in fairly good agreement with the thermocouple temperature (an average difference ∼1 ℃). The present method requires only 15 min of spectral acquisition time for calibration, which is 50 times shorter than that in a recently reported calibration method using the pure rotational Raman spectrum of N2. We envision that it will be an effective asset in Raman thermometry and its applications to cellular thermogenesis and thermoregulation.