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

Abstract

The jellyfish green fluorescent protein (GFP) is a versatile biological marker and reporter. Here we demonstrate that marked polarisation in GFP fluorescence, upon excitation with plane polarised light, can be exploited to enhance the detection of GFP when in the presence of cellular auto-fluorescence and other fluorescent compounds that emit at the same wavelength. The development of flow-though instrumentation dedicated to the sensitive detection of GFP by fluorescence polarisation is described, and used in both a continuous flow and flow-injection format. The intensity, spectral properties and extent of polarisation in the auto-fluorescence of various yeast strains and growth media are investigated. The application of fluorescence polarisation 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.