Hayashi Teruyuki, Fukuda Nanaho, Uchiyama Seiichi, Inada Noriko
The Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma-shi, Nara, Japan.
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
PLoS One. 2015 Feb 18;10(2):e0117677. doi: 10.1371/journal.pone.0117677. eCollection 2015.
Changes in intracellular temperatures reflect the activity of the cell. Thus, the tool to measure intracellular temperatures could provide valuable information about cellular status. We previously reported a method to analyze the intracellular temperature distribution using a fluorescent polymeric thermometer (FPT) in combination with fluorescence lifetime imaging microscopy (FLIM). Intracellular delivery of the FPT used in the previous study required microinjection. We now report a novel FPT that is cell permeable and highly photostable, and we describe the application of this FPT to the imaging of intracellular temperature distributions in various types of mammalian cell lines. This cell-permeable FPT displayed a temperature resolution of 0.05°C to 0.54°C within the range from 28°C to 38°C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature.
细胞内温度的变化反映了细胞的活性。因此，测量细胞内温度的工具可以提供有关细胞状态的有价值信息。我们之前报道了一种结合荧光聚合物温度计（FPT）和荧光寿命成像显微镜（FLIM）来分析细胞内温度分布的方法。之前研究中使用的FPT的细胞内递送需要显微注射。我们现在报道一种新型的可穿透细胞且具有高光稳定性的FPT，并描述了这种FPT在各种类型哺乳动物细胞系中对细胞内温度分布成像的应用。这种可穿透细胞的FPT在HeLa细胞提取物中，在28°C至38°C范围内显示出0.05°C至0.54°C的温度分辨率。使用我们优化的方案，这种可穿透细胞的FPT在孵育10分钟内自发扩散到HeLa细胞中，并且在数小时的观察中表现出最小的毒性。FLIM分析证实了细胞核与细胞质之间的温度差异以及线粒体附近的产热情况，这也是之前使用显微注射的FPT检测到的。我们还表明，这种可穿透细胞的FPT方案可以应用于其他哺乳动物细胞系，COS7和NIH/3T3细胞。因此，这种可穿透细胞的FPT是一种有前景的工具，可用于研究与温度相关的细胞状态和功能。
