Wazawa Tetsuichi, Ozaki-Noma Ryohei, Kai Lu, Fukushima Shun-Ichi, Matsuda Tomoki, Nagai Takeharu
SANKEN (The Institute of Scientific and Industrial Research), Osaka University, Ibaraki, Osaka 567-0047, Japan.
Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.
Biophys Physicobiol. 2025 Apr 8;22(2):e220008. doi: 10.2142/biophysico.bppb-v22.0008. eCollection 2025.
Temperature crucially affects molecular processes in living organisms and thus it is one of the vital physical parameters for life. To investigate how temperature is biologically maintained and regulated and its biological impact on organisms, it is essential to measure the spatial distribution and/or temporal changes of temperature across different biological scales, from whole organism to subcellular structures. Fluorescent nanothermometers have been developed as probes for temperature measurement by fluorescence microscopy for applications in microscopic scales where macroscopic temperature sensors are inaccessible, such as embryos, tissues, cells, and organelles. Although fluorescent nanothermometers have been developed from various materials, fluorescent protein-based ones are especially of interest because they can be introduced into cells as the transgenes for expression with or without specific localization, making them suitable for less-invasive temperature observation in living biological samples. In this article, we review protein-based fluorescent nanothermometers also known as genetically-encoded temperature indicators (GETIs), covering most published GETIs, for developers, users, and researchers in thermal biology as well as interested readers. We provide overviews of the temperature sensing mechanisms and measurement methods of these protein-based fluorescent nanothermometers. We then outline key information for GETI development, focusing on unique protein engineering techniques and building blocks distinct to GETIs, unlike other fluorescent nanothermometers. Furthermore, we propose several standards for the characterization of GETIs. Additionally, we explore various issues and offer perspectives in the field of thermal biology.
温度对生物体内的分子过程有着至关重要的影响，因此它是生命的重要物理参数之一。为了研究温度在生物学上是如何维持和调节的，以及它对生物体的生物学影响，测量从整个生物体到亚细胞结构等不同生物尺度上温度的空间分布和/或时间变化至关重要。荧光纳米温度计已被开发用作通过荧光显微镜进行温度测量的探针，用于宏观温度传感器无法触及的微观尺度应用，如胚胎、组织、细胞和细胞器。尽管已经从各种材料开发出了荧光纳米温度计，但基于荧光蛋白的温度计尤其令人感兴趣，因为它们可以作为转基因引入细胞进行表达，有无特定定位均可，这使得它们适用于对活生物样本进行微创温度观察。在本文中，我们综述了基于蛋白的荧光纳米温度计，也称为基因编码温度指示剂（GETIs），涵盖了大多数已发表的GETIs，面向热生物学领域的开发者、使用者和研究人员以及感兴趣的读者。我们概述了这些基于蛋白的荧光纳米温度计的温度传感机制和测量方法。然后，我们概述了GETI开发的关键信息，重点关注与其他荧光纳米温度计不同的GETIs独特的蛋白质工程技术和构建模块。此外，我们提出了几种GETIs表征的标准。此外，我们探讨了热生物学领域的各种问题并提供了观点。
