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. © 2012 Bogdanov et al.