Department of Chemistry, Kwansei Gakuin University, Sanda, Japan.
Appl Spectrosc. 2020 Oct;74(10):1295-1296. doi: 10.1177/0003702820933908. Epub 2020 Jul 10.
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 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 N. We envision that it will be an effective asset in Raman thermometry and its applications to cellular thermogenesis and thermoregulation.
这里介绍了一种用于校准低频 Raman 光谱中反斯托克斯-斯托克斯强度比的简便实用方法，该方法专门用于测量细胞内的温度。该方法使用液态水的低频 Raman 光谱作为强度标准，水是细胞的主要分子成分，其温度可以通过热电偶独立测量。我们不是直接校准像素强度，而是在 20-200 cm 区域的每个 Raman 位移处通过将根据已知温度的玻尔兹曼因子理论计算得到的反斯托克斯-斯托克斯强度比除以实验得到的强度比来获得校正因子。通过测量其他温度下的水来验证所得到的校正曲线的有效性。经过校准的反斯托克斯-斯托克斯强度比与玻尔兹曼因子的拟合度很好，误差在 1%以内，并且与热电偶温度（平均差异约为 1℃）相当吻合。与使用 N 的纯旋转 Raman 光谱的最近报道的校准方法相比，本方法仅需 15 分钟的光谱采集时间即可进行校准，这比该方法快了 50 倍。我们预计，该方法将成为 Raman 测温及其在细胞产热和体温调节中的应用的有效手段。
