Anti-fading media for live cell GFP imaging

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 p...

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Bibliographic Details
Published in:PloS one 2012-12, Vol.7 (12), p.e53004-e53004
Main Authors: Bogdanov, Alexey M, Kudryavtseva, Elena I, Lukyanov, Konstantin A
Format: Article
Language:English
Subjects:
Antioxidants
Antioxidants (Nutrients)
Biology
Cell cycle
Cells - cytology
Cells - metabolism
Cells - ultrastructure
Cells, Cultured
Chemistry
Culture Media - chemistry
Culture Media - pharmacology
Experiments
Flavonoids
Fluorescence
Fluorescence microscopy
Formulations
Green fluorescent protein
Green Fluorescent Proteins - analysis
HEK293 Cells
Humans
Imaging
Imaging systems
Localization
Microscopy
Microscopy, Fluorescence - methods
Organisms, Genetically Modified
Photobleaching
Photobleaching - drug effects
Physiology
Proteins
Riboflavin
Rutin
Single-Cell Analysis - methods
Vitamin B
Vitamins
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0053004