Subdiffraction imaging through the selective donut-mode depletion of thermally stable photoswitchable fluorophores: Numerical analysis and application to the fluorescent protein Dronpa
Subdiffraction imaging through the selective donut-mode depletion of thermally stable photoswitchable fluorophores: Numerical analysis and application to the fluorescent protein Dronpa
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DOI：
10.1021/ja076128z
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发表时间：
2007-12-26
影响因子：
15
通讯作者：
Hofkens, Johan
中科院分区：
文献类型：
--
作者：
Dedecker, Peter;Hotta, Jun-Ichi;Hofkens, Johan
GFP样荧光蛋白Dronpa的荧光发射的快速和可逆的开/关切换在亚衍射成像中的应用引起了相当大的兴趣。在本文中，我们研究了使用一个甜甜圈模式的光束结合两个以上的重叠激光束，通过选择性切换到非荧光光开关状态，以增加成像分辨率。我们设计并运行了一系列的数值模拟，以确定合适的物理参数的前瞻性，热稳定的光开关分子，在光开关量子产率，抗疲劳性，和可能存在的瞬态非荧光状态。我们的许多发现适用于其他测量，利用甜甜圈光束，这些准则可以用于合成和筛选新的pholoswitchable化合物。我们通过实验证明了通过使用高效和热稳定的Dronpa光开关，使用通常可用的设备来获得更高分辨率的可能性。
The fast and reversible on/off switching of the fluorescence emission of the GFP-like fluorescent protein Dronpa has attracted considerable interest for applications in subdiffraction imaging. In this paper we study the use of a donut-mode beam in combination with two more overlapping laser beams to increase the imaging resolution through selective switching to the nonfluorescent photoswitched state. We devise and run a series of numerical simulations to determine suitable photophysical parameters of prospective, thermally stable photoswitchable molecules, in terms of photoswitching quantum yields, fatigue resistance, and possible presence of transient nonfluorescent states. Many of our findings are applicable to other measurements that make use of donut beams, and these guidelines can be used in the synthesis and screening of novel pholoswitchable compounds. We experimentally demonstrate the possibility of obtaining increased resolution by making use of the efficient and thermally stable Dronpa photoswitching, using equipment that is commonly available.