Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
PLoS One. 2012;7(12):e53004. doi: 10.1371/journal.pone.0053004. Epub 2012 Dec 21.
Photostability is one of the most important characteristic of a dye for fluorescence microscopy. Recently we demonstrated that vitamins present in imaging media dramatically accelerate photobleaching of Enhanced Green Fluorescent Protein (EGFP) and many other green fluorescent and photoactivatable proteins. Here we tested all vitamins of commonly used media (such as Dulbecco's Modified Eagle Medium, DMEM) one-by-one and found that only two vitamins, riboflavin and pyridoxal, decrease photostability of EGFP. Thus, DMEM without riboflavin and pyridoxal can be used as an imaging medium, which ensures high photostability of GFPs at the expense of minimal biochemical disturbance. Then, we tested some antioxidants and found that a plant flavonoid rutin greatly enhances photostability of EGFP during live cell microscopy. In complete DMEM, rutin increased EGFP photostability up to the level of vitamin-depleted DMEM. Moreover, being added to vitamin-depleted DMEM, rutin was able to further suppress EGFP photobleaching. Potentially, new medium formulations can be widely used for fluorescence microscopy of GFP-expressing cells and model multicellular organisms in a variety of imaging applications, where photostability represents a challenge.
光稳定性是荧光显微镜用染料最重要的特性之一。最近我们发现，成像介质中存在的维生素会显著加速增强型绿色荧光蛋白（EGFP）和许多其他绿色荧光蛋白和光激活蛋白的光漂白。在这里，我们逐个测试了常用介质（如 Dulbecco's Modified Eagle Medium，DMEM）中的所有维生素，发现只有两种维生素，核黄素和吡哆醛，会降低 EGFP 的光稳定性。因此，不含核黄素和吡哆醛的 DMEM 可以用作成像介质，这可以确保 GFP 具有很高的光稳定性，同时对生化干扰最小。然后，我们测试了一些抗氧化剂，发现植物类黄酮芦丁在活细胞显微镜下大大增强了 EGFP 的光稳定性。在完整的 DMEM 中，芦丁使 EGFP 的光稳定性提高到维生素耗尽的 DMEM 水平。此外，芦丁添加到维生素耗尽的 DMEM 中，可以进一步抑制 EGFP 的漂白。潜在地，新的培养基配方可以广泛用于 GFP 表达细胞的荧光显微镜和多种成像应用中的模式多细胞生物，其中光稳定性是一个挑战。
