Reversibly photoswitchable fluorescent proteins (RSFPs) are a class of fluorescent proteins whose fluorescence can be turned on and off by light irradiation. RSFPs have become essential tools for super-resolution (SR) imaging. Because most SR imaging techniques require high-power-density illumination, mitigating phototoxicity in cells due to intense light irradiation has been a challenge. Although we previously developed an RSFP named Kohinoor to achieve SR imaging with low phototoxicity, the photoproperties were insufficient to move a step further to explore the cellular dynamics by SR imaging. Here, we show an improved version of RSFP, Kohinoor2.0, which is suitable for SR imaging of cellular processes. Kohinoor2.0 shows a 2.6-fold higher fluorescence intensity, 2.5-fold faster chromophore maturation and 1.5-fold faster off-switching than Kohinoor. The analysis of the pH dependence of the visible absorption band revealed that Kohinoor2.0 and Kohinoor were in equilibria among multiple fluorescently bright and dark states, with the mutations introduced into Kohinoor2.0 bringing about a higher stabilization of the fluorescently bright states compared to Kohinoor. Using Kohinoor2.0 with our SR imaging technique, super-resolution polarization demodulation/on-state polarization angle narrowing, we conducted 4-h time-lapse SR imaging of an actin filament network in mammalian cells with a total acquisition time of 480 s without a noticeable indication of phototoxicity. Furthermore, we demonstrated the SR imaging of mitochondria dynamics at a time resolution of 0.5 s, in which the fusion and fission processes were clearly visualized. Thus, Kohinoor2.0 is shown to be an invaluable RSFP for the SR imaging of cellular dynamics.

中文翻译：

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用于小时延时和亚秒级分辨率超分辨率成像的可光切换荧光蛋白

可逆光开关荧光蛋白（RSFPs）是一类荧光蛋白，其荧光可以通过光照射开启和关闭。RSFP 已成为超分辨率 (SR) 成像的重要工具。由于大多数 SR 成像技术需要高功率密度的照明，因此减轻由于强光照射导致的细胞光毒性一直是一个挑战。尽管我们之前开发了一种名为 Kohinoor 的 RSFP 来实现具有低光毒性的 SR 成像，但其光性能不足以进一步通过 SR 成像探索细胞动力学。在这里，我们展示了 RSFP 的改进版本 Kohinoor2.0，它适用于细胞过程的 SR 成像。Kohinoor2.0 的荧光强度提高了 2.6 倍，发色团的成熟速度提高了 2.5 倍，1。断开速度比 Kohinoor 快 5 倍。对可见吸收带的 pH 依赖性的分析表明，Kohinoor2.0 和 Kohinoor 在多个荧光亮态和暗态之间处于平衡状态，与 Kohinoor 相比，引入 Kohinoor2.0 的突变导致荧光亮态的稳定性更高. 使用 Kohinoor2.0 和我们的 SR 成像技术、超分辨率偏振解调/通态偏振角变窄，我们对哺乳动物细胞中的肌动蛋白丝网络进行了 4 小时的延时 SR 成像，总采集时间为 480 秒，无明显的光毒性迹象。此外，我们以 0.5 秒的时间分辨率展示了线粒体动力学的 SR 成像，其中融合和裂变过程清晰可见。因此，Kohinoor2。