Cell Function and Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan.
Photochem Photobiol Sci. 2010 Feb;9(2):239-48. doi: 10.1039/b9pp00124g. Epub 2010 Jan 18.
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.
光致变色荧光团在超分辨率荧光显微镜技术中起着至关重要的作用，包括光激活定位显微镜（PALM）等技术。PALM 测量产生的图像精度的一个决定性因素是可以从荧光团中检测到的光子数。Dronpa 是一种可还原光致变色的荧光蛋白，已成功应用于 PALM 实验。Dronpa 每个开关循环可获取的光子数取决于其关闭开关的速度，限制了可记录的光子数量。在这项研究中，我们报告了我们的发现，即 Dronpa 的四聚体祖先 22G 显示出较慢的开关速度，并开发了一种突变体，其开关动力学介于 Dronpa 和 22G 之间。我们表明，光致变色的动力学与蛋白质的自组装密切相关，这支持了我们的观点，即动态灵活性决定了光致变色。同样，我们发现由于较慢开关蛋白质在每个开关循环中发射的光子数量更多，因此可以获得更高分辨率的 PALM 图像。
