A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants
A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants
复制标题
DOI：
10.1021/ja074704l
复制
发表时间：
2007-11-14
影响因子：
15
通讯作者：
Hofkens, Johan
中科院分区：
文献类型：
--
作者：
Flors, Cristina;Hotta, Jun-ichi;Hofkens, Johan
用系综和单分子荧光光谱方法研究了可逆光开关类绿色荧光蛋白Dronpa-2和Dronpa-3的光物理性质和光开关机制，并与前体蛋白Dronpa进行了比较。大量观察到的新突变体对光的更快反应和更快的暗恢复也适用于单分子水平。对单分子痕迹的分析使我们能够提取参与前向和后向切换的通路的效率和速率常数，当我们将突变体与Dronpa进行比较时，我们发现了重要的差异。我们根据发色团在P-CAN提供的蛋白质环境中更高的构象自由度来合理地解释我们的结果。这种对光物理参数的彻底了解使我们能够优化这些光致变色蛋白质基于相机的亚衍射极限成像的采集参数。我们证明了Dronpa及其突变体对于使用普通广场显微镜设备的快速光激活定位显微镜(Palm)是有用的，因为单个荧光蛋白可以被多次定位。我们提出了一种通过同时双色频闪照明来实现快速Palm的新方法。
The photophysical properties and photoswitching scheme of the reversible photoswitchable green fluorescent protein-like fluorescent proteins Dronpa-2 and Dronpa-3 were investigated by means of ensemble and single-molecule fluorescence spectroscopy and compared to those of the precursor protein Dronpa. The faster response to light and the faster dark recovery of the new mutants observed in bulk also hold at the single-molecule level. Analysis of the single-molecule traces allows us to extract the efficiencies and rate constants of the pathways involved in the forward and backward switching, and we find important differences when comparing the mutants to Dronpa. We rationalize our results in terms of a higher conformational freedom of the chromophore in the protein environment provided by the P-can. This thorough understanding of the photophysical parameters has allowed us to optimize the acquisition parameters for camera-based sub-diffraction-limit imaging with these photochromic proteins. We show that Dronpa and its mutants are useful for fast photoactivation-localization microscopy (PALM) using common wide-field microscopy equipment, as individual fluorescent proteins can be localized several times. We provide a new approach to achieve fast PALM by introducing simultaneous two-color stroboscopic illumination.