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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Author(s)

    • 岡部 弘基 OKABE Kohki
    • 東京大学大学院薬学系研究科薬科学専攻|独立行政法人科学技術振興機構さきがけ Graduate School of Pharmaceutical Sciences, the University of Tokyo|JST, PRESTO

Abstract

温度はあらゆる化学反応を支配する物理量であり,細胞内温度は細胞内分子の動態や機能に強く影響している.またがん細胞などの病態細胞では亢進した熱発生があることが報告されている1).このことから,細胞内の温度計測により,細胞機能に関する理解が深まるとともに,新規診断,治療法の開発にも貢献すると期待されている.著者は,光を用いた高感度検出を可能とする光学顕微鏡を用いて細胞内温度を高感度かつ定量的に捉える方法を開発してきた.温度変化に鋭敏な応答を示す蛍光性ポリマー温度センサーを高感度イメージング法により検出することで,これまでにいずれも初となる単一生細胞の温度変化の追跡法や,生細胞内の温度分布を可視化する方法を開発した.これらを用いた細胞内温度計測は,従来明らかにされてこなかった細胞内温度に関する興味深い知見をもらしつつあり,生命科学にあまねく貢献できると期待される.

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.

Journal

  • BUNSEKI KAGAKU

    BUNSEKI KAGAKU 63(6), 455-465, 2014

    The Japan Society for Analytical Chemistry

Codes

  • NII Article ID (NAID)
    130004428014
  • NII NACSIS-CAT ID (NCID)
    AN00222633
  • Text Lang
    JPN
  • ISSN
    0525-1931
  • NDL Article ID
    025587341
  • NDL Call No.
    Z17-9
  • Data Source
    NDL  J-STAGE 
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