Fluorescence lifetime imaging and spectroscopy of GFP mutants, and the characterisation of FRET pairs, using high sensitivity, time- and space- correlated single photon counting

• Main Author: Millington, Michael John
• Published: University of Edinburgh 2007
• Subjects: 571.4
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.657807

Fluorescens Lifetime Imaging Microscopy (FLIM) and fluorescence lifetime spectroscopy have been used to characterise the photophysics of different mutants of Green Fluorescent Protein (GFP) and to show evidence of Förster Resonance Energy Transfer (FRET) between different mutants in both model systems and in biologically significant cell samples. Two different imaging systems have been used and compared, a time gated intensified CCD camera (tgiCCD) system and a time and space correlated single photon counting (TSCSPC) system. GFP has been cloned into Escherichia coli and investigated by FLIM and lifetime spectroscopy, both in vivo and in vitro, to show how the fluorescence lifetime of the protein changes as the microenvironment of the protein, and hence the fluorophore, changes. In conjunction with collaborators, cancer cell signalling was investigated by looking for FRET between molecules tagged with different GFPs in fixed mammalian cells. When no consistent energy transfer was observed between GFP and YFP, or between GFP and RFP, in the biologically important cells, a model system was devised.  This model system consisted of the mutant enhanced Cyan Fluorescent Protein (CFP) expressed in fixed cells both alone and as an engineered FRET construct with enhanced Yellow Fluorescent Protein (YFP). This system showed a reproducible FRET signal. Comparison of the results from the CCD camera system, frequently used by biologists to undertake FLIM, and the higher resolution TSCSPC detector highlighted a phenomenon that potentially undermines work that relies on average lifetime results. There is a difference in the rate, or amount, of energy transfer from different conformers of the CFP to the YFP acceptor. This results in an overestimation in the separation distances calculated by FRET between this donor –acceptor pair.