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 (GFP) (S65T) fused to different cellular proteins can easily photoconvert into a red form when cells are grown in media with nonfermentable 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.

Engineered Fluorescent Strains of Cryptococcus neoformans: a Versatile Toolbox for Studies of Host-Pathogen Interactions and Fungal Biology, Including the Viable but Nonculturable State

“…These engineered strains can be used for a wide variety of specific experimental purposes, such as in the assessment of gene expression during infection ( 24 ); analyses of fungal viability ( 25 ), biofilm formation ( 26 ), genome stability ( 27 ), and cell cycle dynamics ( 28 ); quantification of fungal loads in vitro and in vivo ( 25 , 29 , 30 ); and functional studies related to fungal reproduction ( 31 ). Other interesting applications include fluorescent reporters as proxies for environmental conditions such as pH ( 32 ) and carbon source and oxygen availability ( 33 ).…”