Higher resolution in localization microscopy by slower switching of a photochromic protein
Higher resolution in localization microscopy by slower switching of a photochromic protein
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DOI：
10.1039/b9pp00124g
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发表时间：
2010-01-01
影响因子：
3.1
通讯作者：
Miyawaki, Atsushi
中科院分区：
文献类型：
--
作者：
Mizuno, Hideaki;Dedecker, Peter;Miyawaki, Atsushi
光开关荧光团在超分辨率荧光显微镜中起着至关重要的作用，包括诸如光激活定位显微镜（PALM）的技术。PALM测量产生的图像精度的决定因素是可以从荧光团检测到的光子数。Dronpa是一种可逆的光开关荧光蛋白，已成功用于PALM实验。Dronpa每个开关周期可以获取的光子数取决于其关闭开关速率，限制了可以记录的光子数。在这项研究中，我们报告了我们的发现，Dronpa的四聚体祖先，22 G，显示较慢的开关，并开发一个突变体，显示Dronpa和22 G之间的开关动力学。我们发现，光开关的动力学是强烈相关的蛋白质的自关联，支持我们的观点，动态灵活性，确定在光开关。类似地，我们发现，更高分辨率的PALM图像可以与较慢的开关蛋白质，由于其较高的数量发射的光子每个开关周期。
Photoswitchable fluorophores play an essential role in super-resolution fluorescence microscopy, including techniques such as photoactivated localization microscopy (PALM). A determining factor in the precision of the images generated by PALM measurements is the photon numbers that can be detected from the fluorophores. Dronpa is a reversibly photoswitchable fluorescent protein that has been successfully used in PALM experiments. The number of photons per switching cycle that can be acquired for Dronpa depends on its off-switching rate, limiting the number of photons that can be recorded. In this study we report our discovery that the tetrameric ancestor of Dronpa, 22G, shows slower switching, and develop a mutant that displays switching kinetics between those of Dronpa and 22G. We show that the kinetics of the photoswitching are strongly related to self-association of the protein, supporting our view of dynamic flexibility as determining in the photoswitching. Similarly we find that higher-resolution PALM images can be acquired with slower-switching proteins due to their higher number of emitted photons per switching cycle.