Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy

The green fluorescent protein (GFP) has proven to be an excellent fluoresce nt marker for protein expression and localisation in living cells [1-5]. Se veral mutant GFPs with distinct fluorescence excitation and emission spectr a have been engineered for intended use in multi-labelling experiments [6-9 ], Discrimination of these coexpressed GFP variants by wavelength is hamper ed, however, by a high degree of spectral overlap, low quantum efficiencies and extinction coefficients [10], or rapid photobleaching [6], Using fluor escence lifetime imaging microscopy (FLIM) [11-16], four GFP variants were shown to have distinguishable fluorescence lifetimes. Among these was a new variant (YFP5) with spectral characteristics reminiscent of yellow fluores cent protein [8] and a comparatively long fluorescence lifetime. The fluore scence intensities of co-expressed spectrally similar GFP variants (either alone or as fusion proteins) were separated using lifetime images obtained with FLIM at a single excitation wavelength and using a single broad band e mission filter. Fluorescence lifetime imaging opens up an additional spectr oscopic dimension to wavelength through which novel GFP variants can be sel ected to extend the number of protein processes that can be imaged simultan eously in cells. (C) Elsevier Science Ltd ISSN 0960-9822.