Anti-Fading Media for Live Cell GFP Imaging

Abstract

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.

Influence of cell growth conditions and medium composition on EGFP photostability in live cells

“…In particular, F12 contains greatly decreased concentrations of riboflavin and pyridoxine (11- and 67-fold, respectively). These differences appear to be most important, as we previously demonstrated that these vitamins reduce EGFP photostability dramatically (4, 5). …”

“…To improve photostability of fluorescent proteins, site-directed and random mutagenesis have been used extensively (2). As an alternative approach, optimization of imaging medium composition has been suggested recently (4, 5). This approach is based on the fact that GFPs can participate in light-driven redox reactions with intracellular electron acceptors that result in oxidative green-to-red photoconversion (6).…”

“…Therefore, minimally depleted media, which contain all the components of complete media except those that directly decrease EGFP photostability, are preferable for fluorescence microscopy. In particular, media without riboflavin and pyridoxal (both oxidants) and/or containing rutin (an antioxidant) were found to dramatically enhance the photostability of EGFP (4, 5). …”

“…Earlier, we had shown that the positive effect of rutin on EGFP photostability in cells only occurs with freshly prepared water solutions (5). To find appropriate preservation conditions, we tested rutin activity after storage in 2 different solvents, water and DMSO, and at different temperatures (22C, 4C, -20C and -70C).…”

Sensitive detection of active Shiga toxin using low cost CCD based optical detector

“…Mechanistically, Shiga toxin is internalized by the cells via Gb3 receptors and cleaves the N-glycosidic bond of adenine at nucleotide position 4324 in the 28s rRNA of the 60s ribosomal subunit (Endo et al 1998). Elimination of this one adenine residue blocks peptide chain elongation and inhibits protein synthesis.…”