The application of fluorescence polarisation for the enhanced detection ofgreen fluorescent protein (GFP) in the presence of cellular auto-fluorescence and other green fluorescent compounds

The jellyfish green fluorescent protein (GFP) is a versatile biological mar ker and reporter. Here we demonstrate that marked polarisation in GFP fluor escence, upon excitation with plane polarised light, can be exploited to en hance the detection of GFP when in the presence of cellular auto-fluorescen ce and other fluorescent compounds that emit at the same wavelength. The de velopment of flow-though instrumentation dedicated to the sensitive detecti on of GFP by fluorescence polarisation is described, and used in both a con tinuous flow and flow-injection format. The intensity, spectral properties and extent of polarisation in the auto-fluorescence of various yeast strain s and growth media are investigated. The application of fluorescence polari sation is shown to enhance the measurement of the induction of GFP in yeast cells, genetically modified to produce GFP as an indicator of DNA damage, compared with a conventional fluorescence method.