Genetic Tagging and Imaging of Proteins with iFAST in .

is the most common human fungal pathogen, able to reside in a broad range of niches within the human body. Even though systemic infection is associated with high mortality, the fungus has historically received relatively little attention, resulting in a lack of optimized molecular and fluorescent tools. Over the last decade, some extra focus has been put on the optimization of fluorescent proteins (FPs) of C. albicans. However, as the FPs are GFP-type, they require an aerobic environment and a relatively long period to fully mature. Recently, we have shown the application of a novel type of fluorogen-based FP, with an improved version of fluorescence activating and absorption shifting tag (iFAST), in . Due to the dynamic relation between iFAST and its fluorogens, the system has the advantage of being reversible in terms of fluorescence. Furthermore, the combination of iFAST with different fluorogens results in different spectral and cellular properties, allowing customization of the system. Key features • Genetic integration and tagging with the iFAST tag in • Imaging and localization of a protein of interest tagged with iFAST. • Reversibility of fluorescence with iFAST.