Discrimination of fluorescent signals of interest and auto-fluorescence increases the significance of fluorescence measurements. Auto-fluorescence is observed in cellular backgrounds or when screening compounds that fluoresce themselves. Here, it is shown how fluorescence polarization can be used to distinguish between green fluorescent protein (GFP) and auto-fluorescence, mimicked by fluorescein. As GFP is a rather big molecule with low rotational movement it displays a high polarization whereas the small fluorescein gives a low polarization value.
Yeast cells that express GFP upon activation of DNA repair were challenged with the DNA damaging agent methyl methanesulphonate (MMS). Samples were spiked with fluorescein to mimic auto-fluorescence. Taking into account the depolarized emission light (perpendicular to the excitation) MMS-induced GFP was detected despite the presence of fluorescein which emits at the same wavelength as GFP. Furthermore, the fluorescent compounds Proflavin and Methapyrilene were identified to be genotoxic.