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Reversible photobleaching of enhanced green fluorescent proteins

机译：增强型绿色荧光蛋白的可逆光漂白

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摘要

Color variants of green fluorescent protein (GFP) are increasingly used for multicolor imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP). Here we show that experimental settings commonly used in these imaging experiments may induce an as yet uncharacterized reversible photobleaching of fluorescent proteins, which is more pronounced at acidic pH. Whereas the reversible photobleaching spectrum of eCFP corresponds to its absorption spectrum, reversible photobleaching spectra of yellow variants resemble absorption spectra of their protonated states. Fluorescence intensities recover spontaneously with time constants of 25-58 s. The recovery of eCFP can be further accelerated by illumination. The resulting steady-state fluorescence reflects a variable equilibrium between reversible photobleaching, spontaneous recovery, and light-induced recovery. These processes can cause significant artifacts in commonly applied imaging techniques, photobleach-based FRET determinations, and FRAP assays.
机译：绿色荧光蛋白（GFP）的颜色变体越来越多地用于多色成像，荧光共振能量转移（FRET）和光漂白后的荧光恢复（FRAP）。在这里，我们显示了在这些成像实验中常用的实验设置可能会诱导荧光蛋白的尚未表征的可逆光致漂白，这在酸性pH下更为明显。 eCFP的可逆光漂白光谱与其吸收光谱相对应，而黄色变体的可逆光漂白光谱类似于其质子化态的吸收光谱。荧光强度自发恢复，时间常数为25-58 s。 eCFP的恢复可以通过照明进一步加速。产生的稳态荧光反映了可逆的光漂白，自发恢复和光诱导恢复之间的可变平衡。这些过程可能会在常用的成像技术，基于光漂白的FRET测定和FRAP分析中引起明显的伪影。

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