Raarup Merete K, Fjorback Anja W, Jensen Stig M R, Müller Heidi K, Kjaergaard Maj M, Poulsen Hanne, Wiborg Ove, Nyengaard Jens R
Aarhus University, Stereology and Electron Microscopy Research Laboratory, Histoinformatics and MIND Centres, Ole Worms Alle 1185, DK-8000 Aarhus C., Denmark.
J Biomed Opt. 2009 May-Jun;14(3):034039. doi: 10.1117/1.3103338.
Ongoing research efforts into fluorescent proteins continuously generates new mutation variants, some of which can become photoactivated or photoconverted to a red-shifted color upon intense UV or blue light illumination. We report a built-in propensity for enhanced yellow fluorescent protein (EYFP) to undergo irreversible photoconversion into a cyan fluorescent protein (CFP)-like species upon green-light illumination. The photoconversion is thermally activated, happens mainly in fixed, nonsealed cell samples, and may result in a very bright and relatively photostable CFP-like species. The photoconversion efficiency depends on the sample diffusivity and is much increased in dehydrated, oxygenated samples. Given the large variations in conversion efficiency observed among samples as well as within a sample, photoconversion cannot be appropriately accounted for in the analysis of acceptor photobleaching fluorescence resonance energy transfer (pbFRET) images and should rather be completely avoided. Thus, samples should always be checked and discarded if photoconversion is observed.
对荧光蛋白的持续研究不断产生新的突变变体，其中一些在强烈紫外线或蓝光照射下可被光激活或光转换为红移颜色。我们报告了增强型黄色荧光蛋白（EYFP）在绿光照射下不可逆地光转换为类青色荧光蛋白（CFP）物种的内在倾向。这种光转换是热激活的，主要发生在固定的、未密封的细胞样本中，并且可能产生一种非常明亮且相对光稳定的类CFP物种。光转换效率取决于样品的扩散率，在脱水、充氧的样品中会大大提高。鉴于在样品之间以及样品内部观察到的转换效率存在很大差异，在分析受体光漂白荧光共振能量转移（pbFRET）图像时无法适当地考虑光转换，而应完全避免。因此，如果观察到光转换，应始终检查并丢弃样品。
