蛍光イメージング法による生細胞内の温度計測法の開発と応用 [in Japanese] Development and Application of Thermometry in Living Cells by Fluorescence Imaging [in Japanese]
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Temperature is a fundamental physical quantity that governs every biological reaction within living cells, and the temperature distribution reflects the cellular thermodynamics and function. In medical studies, the cellular pathogenesis of diseases (e.g., cancer) is characterized by extraordinary heat production. Therefore, intracellular temperature imaging of living cells should promote a better understanding of cellular events and the establishment of novel diagnoses and therapies. However, intracellular temperature measurements in living cells have not yet been performed because no thermometry has been available. Here, we present our novel methods for intracellular temperature imaging based on a fluorescent polymeric thermometer and their applications to the monitoring and mapping of the intracellular temperature. We have demonstrated the first intracellular thermometry with a fluorescent nanogel thermometer. The fluorescence response of the thermometer with increasing temperature was independent of the KCl concentration, the environmental pH, or surrounding proteins. Fluorescence imaging of the thermometer in single COS7 cells showed the temperature-dependent response upon heating, which provides the calibration curve for intracellular thermometry. Next, we developed a novel fluorescent polymeric thermometer that could diffuse throughout the cell, and applied it to intracellular temperature mapping, where the fluorescence lifetime of the thermometer was adopted as a temperature-dependent variable. Observations of the fluorescence lifetime of the thermometer in a living cell using fluorescence lifetime imaging microscopy (FLIM) allowed intracellular temperature imaging. The intracellular temperature distribution that we observed indicated that the nucleus and centrosome of a COS7 cell both showed a significantly higher temperature than the cytoplasm, and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle. Finally, intracellular temperature variations induced by FCCP (mitochondria uncoupler) were investigated. Both temperature monitoring and imaging showed that the uncoupling of mitochondria provoked the local temperature increase, suggesting that mitochondria undertakes thermogenesis in living cells. Our findings about intracellular temperature demonstrate an intrinsic connection between the temperature and cell function. Thus, our intracellular temperature imaging has a significant impact on the comprehension of cell function, and will provide insights into the regulatory mechanisms of intracellular signaling.
- BUNSEKI KAGAKU
BUNSEKI KAGAKU 63(6), 455-465, 2014
The Japan Society for Analytical Chemistry