Anti-fading media for live cell GFP imaging

PLoS One. 2012;7(12):e53004. doi: 10.1371/journal.pone.0053004. Epub 2012 Dec 21.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cells / cytology
  • Cells / metabolism
  • Cells / ultrastructure*
  • Cells, Cultured
  • Culture Media / chemistry
  • Culture Media / pharmacology*
  • Green Fluorescent Proteins / analysis*
  • HEK293 Cells
  • Humans
  • Microscopy, Fluorescence / methods*
  • Organisms, Genetically Modified
  • Photobleaching / drug effects*
  • Single-Cell Analysis / methods

Substances

  • Culture Media
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins

Grants and funding

This work was supported by the Dinasty Foundation and Russian Foundation for Basic Research grant 11-04-01861a. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.