Thédié Daniel, Berardozzi Romain, Adam Virgile, Bourgeois Dominique
Institut de Biologie Structurale, CNRS, Université Grenoble Alpes, CEA, IBS, 38044 Grenoble, France.
J Phys Chem Lett. 2017 Sep 21;8(18):4424-4430. doi: 10.1021/acs.jpclett.7b01701. Epub 2017 Sep 5.
Green-to-red photoconvertible fluorescent proteins (PCFPs) such as mEos2 and its derivatives are widely used in PhotoActivated Localization Microscopy (PALM). However, the complex photophysics of these genetically encoded markers complicates the quantitative analysis of PALM data. Here, we show that intense 561 nm light (∼1 kW/cm) typically used to localize single red molecules considerably affects the green-state photophysics of mEos2 by populating at least two reversible dark states. These dark states retard green-to-red photoconversion through a shelving effect, although one of them is rapidly depopulated by 405 nm light illumination. Multiple mEos2 switching and irreversible photobleaching is thus induced by yellow/green and violet photons before green-to-red photoconversion occurs, contributing to explain the apparent limited signaling efficiency of this PCFP. Our data reveals that the photophysics of PCFPs of anthozoan origin is substantially more complex than previously thought, and suggests that intense 561 nm laser light should be used with care, notably for quantitative or fast PALM approaches.
绿色到红色的光转化荧光蛋白（PCFP），如mEos2及其衍生物，在光激活定位显微镜（PALM）中被广泛使用。然而，这些基因编码标记物复杂的光物理性质使PALM数据的定量分析变得复杂。在这里，我们表明，通常用于定位单个红色分子的高强度561nm光（~1kW/cm）通过填充至少两个可逆暗态，极大地影响了mEos2的绿色态光物理性质。这些暗态通过一种搁置效应阻碍绿色到红色的光转化，尽管其中一个暗态会被405nm光照快速清空。因此，在绿色到红色光转化发生之前，黄色/绿色和紫色光子会诱导多次mEos2转换和不可逆光漂白，这有助于解释这种PCFP明显有限的信号效率。我们的数据表明，来自珊瑚虫纲的PCFP的光物理性质比以前认为的要复杂得多，并表明应谨慎使用高强度561nm激光，特别是对于定量或快速PALM方法。
