Real-time intravital imaging of pH variation associated with osteoclast activity
Real-time intravital imaging of pH variation associated with osteoclast activity
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DOI：
10.1038/nchembio.2096
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发表时间：
2016-08-01
影响因子：
14.8
通讯作者：
Kikuchi, Kazuya
中科院分区：
文献类型：
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作者：
Maeda, Hiroki;Kowada, Toshiyuki;Kikuchi, Kazuya
活体成像的双光子激发显微镜（TPEM）已广泛用于可视化细胞功能。然而，通常用于细胞成像的小分子探针（SMPs）不能简单地应用于活体成像，因为将它们输送到目标组织的挑战，以及它们在TPEM成像中不理想的物理化学性质。在这里，我们设计并开发了一种具有活性靶向部分、更高光稳定性和荧光开关的功能性SMP，然后通过将SMP注射到活小鼠体内来成像靶细胞的活性。将合理设计的SMP与荧光蛋白结合作为细胞定位的报告蛋白，可以定量测定破骨细胞的活性，并延时成像其与细胞变形和膜波动变化相关的体内功能。实时成像揭示了体内破骨细胞的异质性行为，并为骨吸收机制提供了见解。
Intravital imaging by two-photon excitation microscopy (TPEM) has been widely used to visualize cell functions. However, small molecular probes (SMPs), commonly used for cell imaging, cannot be simply applied to intravital imaging because of the challenge of delivering them into target tissues, as well as their undesirable physicochemical properties for TPEM imaging. Here, we designed and developed a functional SMP with an active-targeting moiety, higher photostability, and a fluorescence switch and then imaged target cell activity by injecting the SMP into living mice. The combination of the rationally designed SMP with a fluorescent protein as a reporter of cell localization enabled quantitation of osteoclast activity and time-lapse imaging of its in vivo function associated with changes in cell deformation and membrane fluctuations. Real-time imaging revealed heterogenic behaviors of osteoclasts in vivo and provided insights into the mechanism of bone resorption.