Modulation of green to red photoconversion of GFP during fluorescent microscopy by carbon source and oxygen availability

Abstract

Studies have reported on the ability of green fluorescent proteins to photoconvert into a red fluorescent form under various conditions, such as the presence of oxidants, hypoxia, as well as under benign conditions using irradiation with a 405 nm laser. Here we show that in Saccharomyces cerevisiae yeast green fluorescent protein (S65T) fused to different cellular proteins can easily photoconvert into a red form when cells are grown in media with non‐fermentable carbon sources. This photoconversion occurs during standard microscopy between glass slide and coverslip, but is completely prevented by imaging on pads of solid medium or in a large volume of medium on an inverted microscope. The observed effect was due to rapid hypoxia of cells with respiratory metabolism in standard conditions for upright microscopy. Photoconversion could be prevented by antioxidant treatment, suggesting that it proceeds via the effects of reactive oxidative species emerging in response to oxygen deficiency. Our results show the need for caution during upright microscopy imaging in conditions where there is active respiration, and demonstrate simple approaches to prevent unwanted GFP photoconversion. They also provide easy means of performing photoconversion experiments on existing GFP‐bearing cell lines, at least in the case of yeast.