Multiple-probe fluorescence imaging applications demand an ever-incrasing number of resolvable probes, and the use of fluorophores with resolvable fluorescence lifetimes can help meet this demand. Green fluorescent protein (GFP) and its variants have been widely used in spectrally-resolved multi-probe imaging, but as yet there have not been a systematic set of mutants generated with resolvable lifetimes. This inventions has utilized error-prone PCR and fluorescence lifetime imaging to screen for mutants of UV-excited green fluorescent protein (GFPuv) the result of which are GFPuv mutants displaying at least 3 distinctly different lifetimes in the range of 1.9ns-2.8ns. These GFPuv mutants have applications in simultaneous imaging and quantification as shown by the aility to quantify the compostion of binary mixtures in time-resolved images using a single detector channel. Also disclosed is the application of the screening method for generating lifetime mutants of other fluorescent proteins.
|Published - Aug 24 2005